THE KINETIC CHARACTERIZATION OF HALOARCULA MARISMORTUI ACYL PHOSPHATASE WITH A NOVEL SUBSTRATE by Aroura Gagnon BSc., University of Northern British Columbia, 2015 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE IN BIOCHEMISTRY UNIVERSITY OF NORTHERN BRITISH COLUMBIA June 2017 ãAroura Gagnon, 2017 Abstract Haloarcula marismortui is a red pigmented halophilic archaeal species, originally derived from the Dead Sea, that grows optimally in temperatures ranging from 40 -50 ºC and NaCl salt concentrations of 0.3 -4 M. Acyl phosphatase (AcPase) is a highly ubiquitous enzyme of ~10 kDa and the smallest known enzyme to hydrolyze carboxy-phosphate bonds despite the unknown physiological substrate. This study characterizes AcPase activity with 2-methoxy benzoyl phosphate (2mBzP) and 4-methoxy benzoyl phosphate (4mBzP), which were synthesized by a synthesis method that was revised from a 1995 study conducted by Paoli et al. The optimum substrate 4mBzP had a kcat of 0.176 sec-1 and a Km of 4.53 mM, while 2mBzP had a kcat of 0.114 sec-1 and a Km of 4.12 mM. AcPase activity with 4mBzP and 2mBzP suggests the physiological substrate contains an electron- withdrawing group that interacts with AcPase. The cooperative nature of AcPase activity with carbamoyl phosphate suggests a new functional range for this enzyme and further characterizes both the enzyme and native substrate. ii Table of Contents Abstract ii Table of Contents iii Table of Figures v List of Tables vi List of Abbreviations vii Acknowledgements ix Chapter 1 Introduction 1 Chapter 2 Literature Review 4 Introduction of Haloarcula marismortui Introduction of Archaea 4 4 Haloarchaea as a Model Organism 5 Haloarcula marismortui 8 Har. marismortui Metabolism Considerations of Metalloproteins 10 Introduction of Acyl phosphatase Acyl Phosphatase Structure 12 13 Acyl Phosphatase Function 16 Introduction of Kinetics Previous Kinetic Studies 19 20 Hydrogenase Maturation Factor, HypF 22 Chapter 3 Materials and Methods 23 Introduction 23 Preparation of the 1167pET21b Vector Ligation 23 23 Transformation 24 Verification of 1167pET21b Sequence 25 AcPase Expression and Purification Expression 25 25 Lysis 26 AcPase Purification 26 Identification of Purified AcPase Protein 27 iii Substrate Synthesis Micro-Synthesis 28 28 Macro-Synthesis 28 Verification of Substrates 29 Purification 29 AcPase Kinetic Assays BzP, 2mBzP, and 4mBzP 30 30 Reversible Reaction 31 Acetate Inhibition 31 Carbamoyl phosphate Activity 31 Chapter 4 Kinetic Investigation 33 Introduction 33 Acyl phosphatase Cloning, Expression, and Purification 35 Substrate Synthesis Micro-Synthesis 42 43 Macro Synthesis 50 Kinetic Activity BzP Activity Assay 61 61 2mBzP Activity Assay 62 4mBzP Activity Assay 63 BzP, 2mBzP and 4mBzP Comparison 64 Acetate Inhibition of AcPase 69 AcPase Reversibility Investigation 70 Carbamoyl phosphate Activity Investigation 71 Chapter 5 Conclusions and Further Studies 73 References 77 Appendix 83 iv Table of Figures Figure 1.1: Modeled structure of acyl phosphatase 1 Figure 2.1: Carboxy-phosphate bond hydrolysis mechanism 13 Figure 2.2: Acyl phosphatase active site 14 Figure 2.3: RRM-fold illustration 15 Figure 2.4: Proposed acyl phosphatase catalytic mechanism 16 Figure 2.5: Carbamoyl phosphate HypF-N reaction 22 Figure 3.1: Synthesis mechanism 29 Figure 4.1: Methoxy-benzoyl phosphate synthesis products 34 Figure 4.2: 1167pET21b cPCR agarose gel 35 Figure 4.3: His- trap acyl phosphatase purification chromatogram 37 Figure 4.4: SDS- PAGE His- trap AcPase purification visualization 38 Figure 4.5: Sizing FPLC chromatogram of acyl phosphatase 40 Figure 4.6: SDS- PAGE sizing visualization 41 Figure 4.7: HPLC Spectra 2mBzP 278nm 44 Figure 4.8: HPLC Spectra 2mBzP 258nm 45 Figure 4.9: Methoxy-benzoic acid standard curves 48 Figure 4.10: Micro-synthesis malachite green assay 49 Figure 4.11: Pyridine standard curve 52 Figure 4.12: Macro-synthesis malachite green assay 53 Figure 4.13: 2mBzP FPLC purification 55 Figure 4.14: 4mBzP FPLC purification 57 Figure 4.15: 2mBzP, 4mBzP retention time comparison 58 Figure 4.16: 2mBzP, 4mBzP wavescans 60 Figure 4.17: Acyl phosphatase BzP Michalis-Menten plot 62 Figure 4.18: Acyl phosphatase 2mBzP Michalis-Menten plot 63 Figure 4.19: Acyl phosphatase 4mBzP Michalis-Menten plot 64 Figure 4.20: Acyl phosphatase acetate inhibition Lineweaver-Burk plot 69 Figure 4.21: Acyl phosphatase reverse activity 70 Figure 4.22: Acyl phosphatase carbamoyl phosphate activity 72 v List of Tables Table 3.1: Primer list 24 Table 4.1: 278nm HPLC data 44 Table 4.2: 258nm HPLC data 46 Table 4.3: Initial synthetic method comparison 50 Table 4.4: Final synthetic method comparison 59 Table 4.5: Kinetic comparison of BzP, 2mBzP, and 4mBzP 65 vi List of Abbreviations AcPase – Acetyl phosphatase BzP – Benzoyl phosphate BzP – Benzoyl phosphate cPCR – Colony polymerase chain reaction CT – Common type CV – Column volume ED – Entner Doudroff pathway EDTA – Ethylene- diamine- tetra- acetic acid FPLC – Fast paced liquid chromatography HPLC – High- pressure liquid chromatography HypF-N – N terminus of the HypF protein ID – Identification kcat – Catalytic constant KDG – 2- keto- 3- deoxy gluconate KDPG – 2- keto-3- deoxy- 6- phosphogluconate Km – Michealis-Menten constant LB – Luria-Bertani medium MT – Muscle type NDP – Nucleotide diphosphate NMP – Nucleotide monophosphate NTP – Nucleotide triphosphate Pi – Inorganic phosphate pI – Isoelectric point ppED – Partial phosphorylative Entner Doudroff pathway RRM – RNA recognition motif rrnAC1167—Haloarcula marismortui acyl phosphatase gene RT – Retention time SDS-PAGE – Sodium dodecyl sulfate polyacrylamide gel electrophoresis TAE – Tris, acetic acid, EDTA buffer TCA – Tri-citric acid cycle TEA – Triethylamine THF – Tetrahydrofuran Tris – Tris(hydroxymethyl)aminomethane Vmax – Maximum velocity 2mBzCl – 2- methoxy-benzoyl chloride 2mBzP – 2- methoxy-benzoyl phosphate 4mBzCl – 4- methoxy-benzoyl chloride vii 4mBzP – 4- methoxy-benzoyl phosphate viii Acknowledgements I could write something all about how growing up I was taught that science was for boys and girls do the boring things like cooking and cleaning and being the pink power ranger (all I wanted was to be the green one but noooo I had to be the pink one), but that’s not quite the case. I grew up being told I could do whatever I wanted, just as long as it made me happy, be that Nancy Drew, a lawyer, a doctor, and even a pop star, so I chose to be a scientist. I really should have gone for Sith Lord, they have cooler uniforms and cookies. And to that is all thanks to my mom and dad (they never did mention that the Sith Lords have way cooler outfits). So, a huge shout out to my parents, who have this magical never-ending confidence in me, that it's mind blowing (even bigger than the face of Boe bombshell). You think I’m kidding, well riddle me this. The day I told my mom that I got into the Master's program here at UNBC she didn't have the usual mom responses instead she goes "well duh I already knew that." When I asked her how she shrugged and said I always knew you would. She never had a doubt and if she did she hid it well. So, no matter what, she had faith in me even when I was pondering giving up and going to clown school, she knew that I would finish, and when I clued in that I actually was going to finish, she gave me a kernel of wisdom for my Defense, "If you puke just keep going no one will notice and if they do you will be even more Hard-Core." And with I have one less thing to worry about any major presentation (and it is advice that I share with anyone nervous it helps). Everyone says my mom is the best, and I always say I know, because she is. I am endlessly thankful for my Supervisor Andrea, I had no idea she would ever want to take me on as a master’s student, so when she said sure I had no idea what to say. And since then I have learned more than I thought I would, one of the things that I have learned is that I am dead certain she is a Wizard and she secretly attended Hogwarts (I am even more certain of this than the Brent Murray is a Vampire thing from the second year). I want to thank her for dealing with my questions (even the stupid ones), and the many texts asking if she's in her office so I don't waste a trip up the stairs, and for just being awesome. Throughout my Master's she was an endless wealth of information and lab tricks and I truly hoped I learned most of them (after all who doesn't love magic tricks). I want the world to know the hard-core beast that my computer is for still carrying on strong through a second university degree. It was with me through last minute papers, allnighters (no regrets), the days that I got nothing done (no shame… lie lots of shame), it traveled ix to and from school and dealt with the day I brought it and forgot it's buddy the cord (or the inverse). Through thick and thin my computer was the Sam of my story and I have learned you should never leave home without a Sam. My computer could have died at any moment and it didn’t, making it my hero (because there is no way I could replace it nor would I have known what to do with its computer corpse). To my computer, I say you did me a real solid and I owe you one, so when word throws a hissy fit (we both know it will) I’ll do you the solid by not throwing you out the window. To my pain in the ass bat crap crazy cat… well you didn't really make things easier with your many nights of singing the song of your people, your fears of death if the door remains closed, your lack of fear in cucumbers, you abundance of trust in insects that turned to fear if they had wings, needing to be one me only when I'm working and being too cat to care when I wasn't, for stealing my spot 2 seconds after I get up (I watched you jump up and lay down you ass), for hating kisses, for being scared of bananas and the smell mint, for being scared of people but obsessed with people watching, for just being you, and lastly the look of shock when I hit you with my Nerf gun (I lie, that does make me feel better!). And lastly, to my Grandpa for tolerating living with a hermit since 2012, and dealing with the aforementioned cat, my 2 am snack sessions, the odd hours that I come home, the strange science talk that slips out, and the sometimes E. coli smell that I bring home. I promise to remember to check my battery whenever I have internet problems and use the infinite wisdom I have learned from your crime shows (that is if I’m going to do it don’t get caught, right?) And a note on UNBC changing dates on my degrees. When I finished my undergrad in December 2014 I did exactly what I set out to do (partially because I was told it was impossible) I did my degree in 4 years. UNBC decided to change that very crucial date, to May 2015 (convocation) a much less of an impressive feat. Let the record show I did finish in 2014, and I finished my master’s in 2017 (not 2018 like my degree will most likely say). The most frustrating thing since reluctant E. coli colonies. Seriously. x Chapter 1 Introduction The speculations of the acyl phosphatase (AcPase) physiological function are centered around the known chemical function of hydrolyzing carboxy-phosphate bonds and the prevalence of this bond in cellular molecules. The aim of this study was to provide insight into a native substrate of Haloarcula marismortui AcPase that may allow the prediction of features of the native substrate within both bacterial and eukaryotic species. This study used a combination of kinetic studies and the investigation of protein complex participation to characterize AcPase activity and to suggest potential native substrates. AcPase is a ubiquitous globular enzyme found across all three domains of life that has no confirmed physiological substrate or function regardless of isozyme species. The characterized chemical function of AcPase is facilitating the hydrolysis of carboxy-phosphate bonds (1). AcPase possesses a compact structure of 2 a helices and a b sheet composed of 5 b strands (figure 1.1) (2). The characterization of AcPase activity is limited to kinetic activity with acyl phosphates and benzoyl phosphate (BzP) (3). The characterization of AcPase is focused on forward reaction direction (hydrolysis of the carboxy-phosphate bond) however, the reverse direction has not been investigated. A known inhibitor of AcPase is inorganic phosphate, a hydrolysis product however, AcPase regulation mechanisms have not been proposed or investigated (3). AcPase shares structural homology and a consensus sequence with Escherichia coli HypF-N terminus facilitates the conversion of carbamoyl phosphate into both carbon monoxide and cyanide ions (4). HypF-N terminus is used to study the mechanism of protein aggregation (5) suggesting AcPase potentially plays a role in aggregation diseases (5). Figure 1.1: The structure of Har. marismortui acyl phosphatase fit to the Pyrococcus horikishii AcPase model PDB ID 2W4D. Shown in ribbon form, N-terminus. The image was produced in Swiss PDB viewer. 1 The kinetic characterization of halophilic enzymes allows for the comparison between halophilic and mesophilic enzymes to determine the impact of salt on enzyme activity rates and enzyme substrate interactions. The majority of halophilic enzymatic activity, however, have not been characterized by kinetic investigations; Har. marismortui acyl phosphatase is no exception. This enzyme has been kinetically characterized in vertebrates (cow, horse, and human are the most common) (1, 2, 3, 6, 7, & 8). Therefore, the characterization of a halophilic isozyme of AcPase is necessary to investigate the impact of elevated salt concentrations on the activity of this enzyme. Har. marismortui is a halophilic archaeal species that grows in a wide range of salt concentrations from, 0.3- 4 M, and mesophilic temperatures (9). Arising from the Dead Sea, Har. marismortui adapts to the saline environment by sequestering KCl intracellularly to counterbalance the extracellular concentrations of NaCl (10). Intracellular molecules, mainly proteins, are adapted to remain soluble and functional in the intracellular saline environment that Har. marismortui maintains. The first objective of this thesis was to kinetically characterize Har. marismortui AcPase with synthetic benzoyl phosphate-derived substrates to determine how the introduction and placement of an electron-withdrawing group impacts catalytic function. The presence and location of the electron-withdrawing group on the benzoyl group was expected to have different effects on the carboxy-phosphate bond that Har. marismortui AcPase subsequently hydrolyzes. The location of the electron-withdrawing group on the substrate molecule was expected to suggest which position is most favourable leading to the characterization of a potential native substrate. The second objective of this thesis was to use kinetic studies to further characterize the potential function of AcPase by investigating the conditions Har. marismortui AcPase functions in, directionality of this enzyme, and a new potential substrate. The inhibitory effect of acetate was investigated to characterize the best conditions in which to study this enzyme. The investigation of the reversibility of AcPase determined if Har. marismortui AcPase functions optimally in a singular direction or in two directions. Both studies provided additional insight into the potential function of this enzyme. The investigation of Har. marismortui AcPase activity with carbamoyl phosphate determined if AcPase hydrolyzes the phosphate group of carbamoyl phosphate suggesting the potential for Har. marismortui AcPase to possess HypF-N like 2 function. Combined these three studies were expected to broaden the knowledge of AcPase to characterize the function of this enzyme. Any characterizations of both potential native substrates of Har. marismortui AcPase and activity can be inferred for both bacterial and eukaryal species due to the widespread nature of this enzyme. Characterization of Har. marismortui AcPase will aid in the determination of the physiological function of AcPase. 3 Chapter 2 Literature Review Introduction of Haloarcula marismortui Introduction of Archaea All organisms fall into one of the three domains of life, Bacteria, Eukarya or Archaea. Under the classification of Bacteria organisms: are unicellular, lack extensive cellular metabolism and cellular organizational structures, possess minimalistic genetic material stored in circular chromosomes and live in a diverse range of environments with the ability to adapt to changes in their environment (11). Those classified as Eukarya: range from being unicellular to multicellular, contain both internal cellular organization and a sophisticated metabolism, possess an extensive genome that is tightly compacted into chromosomes and are highly adapted to the moderate environments they inhabit (11). Organisms classified as Archaea: are unicellular. contain unique metabolisms with traits, pathways found in both bacterial and eukaryal species, and a blend of both bacterial and eukaryal cellular organizational structures. Archaeal species possess traits between the two extremes of simple and complex organisms, indicating that these organisms share early ancestry with both Bacteria and Eukarya. While found universally, they tend to dominate in the extreme environments they are commonly found inhabiting. The characteristic of containing features that match the two different domains of life makes archaeal species a curious collection of organisms that should be explored in depth. Archaeal organisms were initially unicellular organisms isolated and identified from niches so extreme no life was thought to survive. These extreme niches include those that are: extremely hot, extremely cold, complete darkness, intense sunlight, highly alkaline, highly acidic in nature, and lastly under high levels of pressure (12). These niches are given the title of ‘extreme’ because of the harsh nature, the apparent absence of biological diversity and are usually lethal to organisms adapted to moderate environments (12). Despite the notion that Archaea are only found in extreme environments, they have been known to thrive in moderate conditions (12). Archaeal species can make up 20% of the probiotic mass of marine plankton and they can also play key roles in soil communities. Archaeal species 4 can also be found acting as a symbiont in the digestive tract of eukaryotic animals a lifestyle previously thought to be possessed solely by bacterial species (12). The wide range of environments that archaeal species inhabit demonstrate the physiological diversity of these organisms. The inherent diversity of this domain is highlighted by the following classifications of various archaeal species: methanogenic organisms, sulfate reducers, extremely halophilic, cell-wallless, extreme thermophilic and S0 reducers (12). These classifications are not exclusive and serve as guidelines to classify the diverse organisms found in this large domain. These physiological types are similar to those found when classifying bacterial species, due to the inherent similarity of being unicellular and filling similar niches or roles in the community. Halophilic species are characterized by the highly saline environment that they have adapted to inhabit. Halophilic environments generally are mesophilic, with temperatures ranging from 15- 45 ºC, with salt concentrations that vary in the range of 0.5- 4 M (12) providing these ‘salt-loving' organisms with the potential for a wide range of adaptations to the varying environmental conditions (13). Halophilic archaea generally are chemoorganoheterotrophs, but there are exceptions to this. Halophilic archaea possess a glycosylated surface layer (S-layer) which replacing a cell wall and is capable of staining gram negative or positive. Some halophilic archaeal species have been found to possess either bacteriorhodopsin or halorhodopsin as analogous energy production using a light generated proton gradient producing ATP (12). Haloarchaea as a Model Organism Haloarchaeal organisms are used to investigate the broad processes and pathways necessary to thrive in the highly saline environment. The investigation into these processes are made possible through molecular biology techniques. For example, Haloarchaea were the first type of Archaea to have a successful vector transformation (14). Halophiles must adapt to the fluctuating environmental salt concentration brought about due to either the evaporation or the addition of water to the environment. The common adaptations halophiles possess to counter their harsh environments include: osmoadaptibility, chromosome maintenance, transcriptional regulation, protein export, protein degradation, gas vesicle synthesis, sensing and motility processes. In general, the halophilic environment provides organisms and enzymes a unique set 5 of conditions pushing them to adapt and evolve in unique manners generating novel cellular mechanisms and organisms (14). Haloarchaea, due to the necessity of their shared ‘extreme' environment, possess a collection of traits that set them apart from their mesophilic counterparts. Together the combined traits allow these organisms to be viable in the high saline environment (14). One of these traits is the possession of a genome with a higher G+C content; the average G+C content is 60% giving the genomic DNA a greater number of hydrogen bonds holding the two strands of DNA together which results in an increase in stability. Halophilic species have the capability to sense and react to the fluctuating salt concentrations present in their halophilic environments. This reaction means that these species need to possess a more versatile and flexible metabolism, containing more robust enzymes. Halophiles also tend to possess multiple copies of genes that mesophilic species possess only a single copy. Thermophilic environments require organisms to possess proteins that have higher internal stability (15). Thermophilic proteins increase stability by increasing hydrophobicity, internal salt bridges and hydrogen bonds. The overall structure of mesophilic proteins tends to have an increased in helical structure whereas the overall structures of extreme thermophilic proteins tends to have an increase in b sheet structure. In both mesophilic and extreme thermophilic proteins, polarity increases in regions exposed to the cytosol allowing for more charge-charge interactions (15). The highlighted characteristics emphasis mesophilic protein species as flexible by decreasing rigid intramolecular interactions while increasing weaker intramolecular interactions. Whereas extreme thermophilic protein species are less flexible with increasing quantity of rigid intramolecular interactions by replacing weaker intramolecular interactions. Organisms living in hypertonic environments must live with cellular hypertonic stress, mainly water loss leading to increased extracellular concentrations which hinders the intracellular metabolism (16). Increasing the solute concentration outside of the cell pulls water from inside the cell causing the intracellular and extracellular solute concentrations to equal one another. The resulting water loss decreases cell volume, concentrates intracellular metabolites and produces macromolecular crowding. These changes disrupt homeostasis, biochemical reaction rates, and elevate reactive oxygen levels which strains the cytoskeleton, depolarizes the mitochondria and damages both DNA and proteins. 6 The adaptation used by Har. marismortui to prevent hypertonic cellular stress is the sequestration of KCl salt which counterbalances high osmotic pressures (10). However, halophilic conditions are detrimental to both enzymatic structure and function. Halophilic proteins have increased quantities of intramolecular salt bridges and larger quantities of the carboxylic acid containing residues on their outer surface than their mesophilic counterparts (17). To remain both soluble and functional in high saline environments, halophilic proteins have lower isoelectric point (pI) values than their mesophilic orthologs. Decreasing the lysine residues present contributes to further decrease in pI (10). The outer protein surface is coated in acidic residues which contributes further to decreasing the pI, provides more opportunities for intermolecular interactions, allowing the protein to remain soluble, and prevents self-aggregation (17). The externally positioned acidic residues that are associated with hydrated salt ions act as a solvation layer, a further measure to ensure the protein remains soluble (18). Evolutionary pressure has led to halophilic enzymes possessing fewer hydrophobic residues and positioning the hydrophobic residues present deep inside a hydrophobic core (10). This reduction in external hydrophobic protein surface also reduces the surfaces available for protein-protein interactions, as most protein-protein interactions occur through the interactions of hydrophobic patches on external protein surfaces. Decreased protein-protein interaction sites strengthen those remaining and increases the stability of these interactions. Smaller hydrophobic residues are present at higher frequencies than larger ones which further compacts the hydrophobic core (10). The specialization of halophilic proteins results in peptides that are highly unstable in low salt conditions where they aggregate, cease function or both (19). Because of the adaptations that increase both protein stability and solubility in high saline environments, halophilic proteins fold properly only in halophilic environments (14). The intracellular conditions of haloarchaeal species present a unique set of challenges when working with these organisms. The greatest challenge comes from maintaining the high salt concentrations necessary for native protein function. The natural low pI of halophilic proteins and the high salt concentrations make ion exchange chromatography an ineffective technique for purifying a protein of interest from these organisms (14). 7 Haloarcula marismortui The organism of interest is the red pigmented haloarchaeon that is found in the Dead Sea, Haloarcula marismortui. This species thrives in NaCl concentrations ranging from 0.3- 4 M (9) and grows optimally at moderate temperatures ranging from 40- 50 °C (20). Like most halophilic Archaea this organism is aerobic (21). Har. marismortui is unicellular with irregular flat disk morphology, rod shapes are found sporadically throughout the culture (20), and is motile with a flagellum that is comprised of 2 archaeal flagellin phenotypes (21). As with many archaea, instead of a cell wall there is an S-layer glycoprotein that coats the extracellular surface. The Slayer glycoprotein is a modified N-linked glycan that is similar to that found in Halofax volcanii, even though the two pathways to produce the glycoprotein are completely different (22). The Har. marismortui genome consists of approximately 4275 kb which codes for 4242 proteins and is split up among nine circular chromosomes of varying size (21). Over 58% of this genome encodes for protein products, a larger percentage than that of a typical prokaryotic genome (21). Halophilic genomes possess a general trend in that the larger chromosomes have a higher G+C content of 62.35%, while the smaller chromosomes have a lower average G+C content of 57%. The reasoning behind larger chromosomes in halophilic genomes possessing larger G+C content is still a mystery. Another peculiar trend in the haloarchaeal genome that remains unclear is that at least three of the smaller replicons carry vital genes for survival. The replicon pNG600, for example, contains the only copy of the gene for aconitase, a protein necessary for the citric acid cycle to function. This replicon also carries a dozen cation transport proteins of various specificities, which are all required for the organism’s response to heavy metal stress (21). Like other unicellular species, Har. marismortui has the capability take up genetic material from the extracellular environment with a transformation efficiency of 100 colonies produced from the transformation of 1µg of plasmid, this, however, is not an efficient or common phenomenon (14). When the Har. marismortui chromosome 1 and Halobacterium NRC1 chromosome are compared, they share approximately 65- 70% similarity, showing that they are obvious homologs (21). Other orthologs are distributed among the other chromosomes at a higher frequency than expected if they were placed at random (21). These genetic studies indicate that at one point Har. marismortui and Halobacterium NRC1 had a common ancestor and had since diverged. Due to the dynamic halophilic environment, Har. marismortui is subject to intense UV damage, 8 desiccation, and rehydration. It is thought that this results in DNA damage followed by extensive homologous recombination, large- scale inversions and genomic rearrangements producing two genomes with different organizational structures (21). Har. marismortui adapts to its environment by sequestering potassium ions, requiring intracellular enzymatic pathways to also adapt (10). The glycolytic pathway that Har. marismortui employs is an example of this adaption. The partial phosphorylative EntnerDoudroff pathway (ppED) degrades glucose to pyruvate, a necessary conversion for anabolic pathways (23). The first step in the classic ED pathway is the phosphorylation of glucose producing glucose- 6- phosphate, an absent step in the ppED. The ppED pathway begins with directly converting glucose into gluconate (23). In the classic ED pathway, the conversion of 6phosphogluconate from glucose- 6- phosphate is the second step. Therefore, the first ppED step compacts the first two classic ED pathway steps. The next ppED step, which converts 2- keto- 3deoxy gluconate (KDG) into 2- keto-3 -deoxy- 6- phosphogluconate (KDPG) is absent in the classic ED pathway. KDPG instead is produced by the conversion of 6- phosphogluconate (23). After producing KDPG both pathways operate in the same manner, converting KDPG into both pyruvate and glyceraldehyde- 3- phosphate. In general, the ppED pathway compacts the classic ED pathway, with both fewer enzymes and steps, decreasing the energy required to maintain the pathway. This results in fewer chances for a misfolding or denaturation event causing the pathway to cease function, making this a more efficient version of the classic ED glycolytic pathway. Har. marismortui possesses an extensive metabolism that includes energy production, catabolism of excess nutrients and cycling metabolic nitrogen (21). The modified glycolytic pathway products lead into the TCA (tri-citric acid cycle), producing both energy and metabolic precursors (21). In amino acid synthesis, the Har. marismortui genome codes for 16 proteins that currently are identified. Select amino acids are catabolized for energy, metabolic carbon, and nitrogen. The reverse TCA works in conjunction with phosphoenol pyruvate carboxylase (found in gluconeogenesis) to fix organic sugars (21). Under anaerobic conditions, the arginase pathway degrades arginine forming a unique halophilic metabolic pathway (21). The common anaerobic arginine pathway (found in Halobacterium NRC1) ferments arginine by the enzyme, arginine deiminase. The Har. marismortui degradation of arginine results in both ornithine and urea. The ornithine produced 9 follows one of two paths, it is converted into either arginine or glutamate. The conversion to arginine begins with converting aspartate to fumarate, the remaining conversion steps occur in the arginase pathway, showing the interconnectivity between the arginase pathway and the TCA cycle. The produced urea is then further degraded into both ammonia and carbon dioxide by the multi- subunit enzyme urease. A single round of the urea cycle consumes three molecules of ATP for every aspartate molecule that is metabolized. The main purpose of both the arginase pathway and the urea cycle in Har. marismortui is to convert the excess environmental amino acids from the environment into desired TCA intermediates (21). Halophilic environments are characterized by high salt concentrations, low oxygen solubility and high light intensity requiring inhabitants to utilize the abundance of light present. Organisms that live in these environments often possess opsin proteins which uses the abundance of light and a proton gradient to produce chemical energy (21). The Har. marismortui genome possesses six opsin genes, where four are orthologs of Halobacterium NRC1 opsin genes leaving two with unknown origins and functions that were named xop1 and xop2. The Xop1 protein contains the majority of the bacteriorhodopsin functional residues which suggests that this opsin functions as a second bacteriorhodopsin (21). The inclusion of a second bacteriorhodopsin is speculated to broaden the energy producing spectra of the organism. The function of Xop2 is reminiscent of an ion pump opsin giving Xop2 the potential to function as a photoreceptor. Har. marismortui not only takes advantage of the intense light present, it also senses and responds to changes in its environment. Har. marismortui possesses two sensory pigments Phr1 and Phr2 and both absorb maximally at blue wavelengths. These two pigments proteins possess high amino acid sequence similarity and possess different functions (21). Phr1 functions as a DNA repair photolyase while Phr2 functions as a cryptochrome which mediates cellular responses to blue light. Har. marismortui regulates its metabolism in response to the day/night cycle by using two circadian clock regulator proteins that were identified in its genome (21). Har. marismortui Metabolism Considerations of Metalloproteins It is speculated that nearly half of all proteins are metalloproteins regardless of the organism, and Har. marismortui is no exception. Metalloproteins require at least one transition metal ion to be functional (24). These transition metals include manganese, copper, cobalt, iron, and zinc. Small intracellular concentrations are required for these ions as in high concentrations 10 they are toxic, damaging DNA and impairing membrane function and integrity. Metals that cycle through redox states, and less dynamic metallic ions, produce reactive oxygen species that interact with DNA, proteins and cellular membranes which disrupts the structure and function of all three. The most common metal ions required, manganese and zinc, are the most toxic. Metal toxicity is prevented by maintaining low intracellular metal ion concentrations. However, low concentration requirements differ among organisms. Differing metal ion concentrations are maintained by a variety of methods that differ among organisms (24). These methods include selective uptake of the metal ions by specific membrane transport enzymes, trafficking, extracellular efflux of the metal ions, enzymatic conversion of the metals to less toxic redox states, or sequestering of the metal ions by either ferritins or metallothioneins. The most common method to regulate these mechanisms is intracellular free metal ion concentration. There are a variety of influences impacting an organism's stress response to high metal ion concentrations, including salinity, pH, temperature, and growth medium components (24). The response mechanisms of Haloarchaea to fluctuating metal ion concentrations in their environment are not yet fully understood (24). It is speculated that a decreased metal ion concentration response mechanism occurs through protein-protein interactions between metalloproteins. These interactions are speculated to play a prominent role in metal ion trafficking, more so than metal ion sensors that directly bind and mediate metal ion uptake and efflux. It is suggested that the apoenzymes are inactive in the absence of metal ions due to protein-protein interactions between the apoenzymes not present in the holoenzyme. Binding to metal ions serves to regulate transcription, impacting gene expression. It is still unclear of what genes are impacted and the reasoning for those select genes to be affected. A common mechanism that Haloarchaea employ to withstand elevated metal ion concentrations present in the extracellular environment is a growth arrest phenotype (24). The growth arrest allows the organism to resist the environmental change long enough for the environment to return to more optimal growth conditions. After five hours in elevated metal ion concentrations transcriptional changes affect approximately 2400 genes which is speculated to lead to mRNA changes that cascade into events transforming the cell into a state more suitable for the new environmental condition (24). Copper is one of the essential trace metals found in two oxidation states and possesses a diverse set of roles in enzymatic function including: respiration, oxidative protection, sugar 11 metabolism and aromatic metabolism (13). When extracellular copper concentrations increase, this essential cofactor becomes cytotoxic. Too high of an intracellular copper concentration increases: reactive oxygen species, copper bound lipids, protein thiol groups and disruptions to iron-sulfur clusters (13). Together they work to disrupt DNA structure, protein structure and membrane integrity leading to cellular death. The possibility of cell death requires copper concentrations to be tightly regulated. The most common method to tightly regulate intracellular copper concentrations is to sequestering copper ions and maintaining an active efflux of copper ions out of the cell by copper-transporting ATPase. Har. marismortui possesses a minor cellular mechanism that regulates intracellular copper concentrations that are easily overwhelmed with extracellular concentrations of 2 mM are cytotoxic (13). The lack of response to a mild copper increase is likely due to the absence of fluctuating high copper concentrations present in its environment. Introduction of Acyl phosphatase Har. marismortui possesses both an extreme intracellular environment for protein function and the specialized metabolic network to be functional in this intracellular environment. The investigation of robust enzymes found in halophiles as well as the comparison to their mesophilic counterparts’ can shed light on the effect of the environment on both enzymatic structure and function. An example of how the environment has shaped enzymatic structure is demonstrated in the case of Har. marismortui esterase which is one of the few halophilic enzymes to be fully characterized (25). This esterase isoform has been characterized to be fully functional in the presence of high KCl concentrations and loses all function in the absence of these high KCl concentrations (25). Acyl phosphatase (AcPase) is an enzyme that can be used to study halophilic enzymatic adaptations as this enzyme is found in all domains of life and in nearly every living organism. AcPase is a highly ubiquitous globular enzyme that possesses a primary sequence ranging from 90- 100 amino acids and has an average molecular weight of 10 kDa, placing it on the small scale of proteins as proteins can possess masses of upwards of 200 kDa (16). AcPase is a cytosolic enzyme, though in some eukaryotic species it is also found in the nucleus (2). As with any enzyme, the primary sequence and the overall structure varies depending on the organism it is found in. An example of a unique structural variation is the E. coli AcPase is the only known 12 AcPase isoform that possesses a disulfide bond (26). Mammals possess two different isoforms of AcPase, that share approximately 55% sequence homology (7). These two isoforms are differentially expressed depending on the organism’s tissue. Muscle type (MT) AcPase is expressed mainly in skeletal muscle and heart tissue, while Common type (CT) AcPase is expressed in all tissue types with high expression levels in brain tissue, white blood cells and testicular tissue (3). Both mammalian isoforms share 40% homology with the Drosophila melanogaster AcPase isoform which suggests that the vertebrate genes and invertebrate gene share a common genetic ancestor (27). Acyl phosphatase is the smallest enzyme known to catalyze the hydrolysis of a carboxyphosphate bond (1). The hydrolysis reaction requires a free water molecule to cleave the carboxy-phosphate bond of the substrate molecule (figure 2.1). After cleaving the carboxyphosphate bond inorganic phosphate (Pi) is released which results in a carboxylic acid derivative of the original substrate. There are still several missing pieces in the characterization of AcPase function, which include: the AcPase native substrate (the nature of the R group in figure 2.1), if the removal of the phosphate group is the only chemical transformation that the substrate undergoes and if it is the first step of a series of chemical transformations are unknown. This means that while the enzyme has been well studied in various species, the biological function of AcPase remains a mystery. Figure 2. 1: The general chemical reaction of the hydrolysis of the carboxy-phosphate bond catalyzed by acyl phosphatase. The R group can be replaced by a great number of molecular groups from a benzene ring to an acetate group Acyl Phosphatase Structure The crystal structure of Pyrococcus horikishii AcPase possesses a typical a/b globular protein structure that is seen in a wide range of other globular proteins (PDB ID 2W4D & 28). This crystal structure was used to generate a model of Har. marismortui AcPase structure 13 (figure1.1). This model consists of two a helices that face 4/5 b strands forming an a/b sandwich domain which produces a babbab secondary structure (26). For AcPase, this forms a five stranded slightly twisted anti-parallel b sheet. On one side of the structure there are two a helices while on the other, the b sheet remains completely exposed to the cytosol and is available for protein-protein, protein-nucleic acid, or protein-small molecule interactions (29). This protein sandwich structure forms a pear shape that possesses stabilizing intramolecular forces between the amphipathic a helices, and hydrophobic interactions found in both the a helices and b sheet. The tight packing of the hydrophobic residues between the a helices and b sheet ensures there are no internal cavities within the protein (29). A portion of the AcPase active site falls within a consensus sequence that is found in known AcPase isoforms (figure 2.2). Residues Arg20 and Asn38 are the catalytic residues that make up the active site, in figure 2.2 these residues are dark blue. The majority of the consensus sequence is depicted in brown and these residues form a loop that likely interacts with substrates and brings them to the catalytic residues. This consensus sequence: FGRVQGVXFR, is used to identify new AcPase isoforms and was determined from the amino acid alignment in the appendix figure A1. Figure 2.2: Visual representation of both the AcPase active site (residues in dark blue) and the loop composed of residues of the consensus sequence. Produced in Swiss PDB viewer using PDB ID 2W4D. Modeled from the crystal structure of Pyrococcus horikishii. The AcPase fold falls into the class of common structures throughout biology known as the RNA recognition motif fold (RRM-fold). The RRM-fold, (figure 2.3), consists of two a helices packed against an anti-parallel b sheet resulting in a bilayered structure (30). The resulting compact structure possesses a large surface area exposed to the cytosolic environment, 14 producing a region ideal for protein-protein interactions, protein-nucleic acid interactions, and protein-small molecule interactions which produces a highly interactive protein structure. The catalytic residues of the proteins that possess this structure vary as widely as their functions vary. The multifunctional portion of the RRM-fold is the large exposed area of the b sheet, which possesses the greatest potential for diverse interactions and functions. A wide range of enzymes make use of this structure and a have a variety of positions for their catalytic residues. PSUS, cyclase, polymerase MSOR Primase (polymerase) Cyclase, polymerase AcPase NDPK NH2 COOH Figure 2.3: The basic topography diagram of the RRM- like fold. The positions of different enzyme catalytic residues are indicated by the blue circles (30). Acyl phosphatase Proposed Mechanism Knowledge of the 3D protein structure allows for the enzymatic mechanism of how AcPase facilitates the hydrolysis of a carboxy-phosphate bond to be investigated. A catalytic mechanism was proposed by Stephani et al (1997) from the investigation of the horse MT (muscle type) AcPase active site (figure 2.4) (2). There are two crucial residues for the catalytic activity of AcPase, Arg23 and Asn41. Arg23 acts as the main phosphate binding site whereas Asn41 binds the catalytic water molecule, aligning it in the optimal orientation for hydrolysis. The proposed catalytic mechanism requires a protonated group, later identified (by assessing pKa values) as a lysine residue. The resulting bond between Asn41 and the water molecule activates the water molecule. The activated water molecule then through a nucleophilic mediated attack, forms a penta-coordinated intermediate with the phosphorus atom. This newly formed intermediate is highly unstable and rapidly decomposes releasing both inorganic phosphate (Pi) and the carboxylic acid form of the original substrate. This proposed mechanism requires further studies to be verified. 15 Figure 2.4: The proposed catalytic mechanism of the hydrolysis of a carboxy-phosphate bond facilitated by acyl phosphatase (2) Acyl Phosphatase Function The classic school of thought is that each enzyme possesses a specific structure which impacts its specific function and each enzyme has its own specific substrate(s), AcPase lacks this inherent specificity. AcPase acts upon a variety of substrates, both naturally occurring and synthetic with only apparent defining factor is the presence of a carboxy-phosphate bond. The capability to act upon a range of substrates classifies AcPase as a promiscuous enzyme. In broad terms, promiscuity is divided into a series of categories to define the phenomenon (31). AcPase is classified as being substrate promiscuous; it lacks enough specificity in that it acts on a broad range of substrates in the same manner (31). This broad acceptance of substrates allows the use of non-native substrates for kinetic studies while it also increases the difficulty in determining the physiological function of AcPase. 16 The RRM protein fold found in AcPase is used in a range of enzymatic functions. The most common protein motifs that employ the RRM-like fold are the aspartate kinase, chorismate mutase, and TyrA (ACT) (32) binding domains, metal coordinating ferredoxins, and RNA binding domains (30). The RRM-like fold offers a wide range of functions and reduces the ability for homology studies to resolve physiological functions of proteins that possess this fold. Typically, homology studies compare structures of known proteins to the structures of unknown proteins to gain insights into potential function. In this case, however, homology studies on AcPase are unable to provide insights into the physiological function of AcPase. Despite the understandings of the chemical function of AcPase, there is little known about the physiological function due to the AcPase native substrate being unknown. The first attempt to resolve a potential function of AcPase came from homology studies. These homology studies found that AcPase possesses approximately 13.5% structural homology with RNA binding domains, which increases to 38% when only hydrophobic regions are considered (33). This structural homology suggests that AcPase could function in RNA binding (33). Of the potential RNA binding functions, AcPase was speculated to bind NTPs and NDPs and catalyze the release of Pi by hydrolyzing the phosphate bonds (34). The subsequent investigation into this function revealed the production of NDP's from NTPs by the release of Pi occurred in a time-dependent manner, similar to the ‘ticking time bombs’ nature of GTPases (35). A kinetic investigation revealed that AcPase possesses a Kcat similar to other NTP hydrolyzing enzymes suggesting NTPs are potential native substrates for AcPase (34). The speculated AcPase function of binding to NTP/NDP was further investigated by Paoli et al (2000) through a series of kinetic assays determining the cleavage of NTPs/NDPs producing either NDPs or NMPs, respectively (35). All assays were performed in a 0.1 M sodium acetate buffer and in the absence of Mg2+ ions, as Mg2+ was found to inhibit AcPase mediated hydrolysis. The inhibition of AcPase activity is likely due to the ion's ability to coordinate with phosphate groups stabilizing the NTPs. The metal ion may also form interactions with the active site preventing hydrolysis. The inhibition by Mg2+ was shown in the comparison of assays with Mg2+ and assays when the Mg2+ concentration was halved. The hydrolysis reaction was monitored by assessing aliquots for the cleavage of NTPs or NDPs using HPLC. The study investigating NTP/NDP cleavage by AcPase (35) was unable to definitively show the cleavage of NTPs/NDPs occurred due to AcPase activity rather than due to the absence 17 of Mg2+. The time points that the reaction was monitored at was in minutes rather than seconds, the standard time frame for enzymatic reactions. After 180 minutes, all three NTP, NDP and NMP peaks were identified by the HPLC spectra. The presence of all three at the same time point indicated that there was a mixture of three species present. This suggested the cleavage observed was not due to AcPase activity, but due to the chemical nature of NTPs and NDPs. In the absence of Mg2+, there was nothing present to stabilize these molecules and prevent selfcleavage. It is the combination of both the long-time scale and the absence of Mg2+ that prevents the study from demonstrating that AcPase binds NTPs and facilitates the hydrolysis of the carboxy-phosphate bond. RNA binding was not the only proposed potential function of AcPase due to the chemical transformation that AcPase facilitates. The carboxy-phosphate bond is common in many cellular molecules which offers a new set of potential functions for this enzyme. One widely accepted potential function is the regulation of ion channels (3, 28, 29, 34, &35). Expression levels of AcPase in metastatic cell lines supports that AcPase potentially functions in regulating ion pumps (35). Ion pump control is speculated to occur by the hydrolysis of the aspartyl-phosphate bonds present which controls activity (3). Suggesting that AcPase could act in the manipulation and regulation of intracellular concentrations of ions such as Na+ and Ca2+ (7). ACT domains use the same RRM-fold as AcPase and are commonly involved in binding with small regulatory molecules, such as amino acids which leads to allosteric regulation (36). Highly active tissues with a high functioning glycolytic pathway also possess high AcPase expression levels. Muscles tissues in freshly hatched chickens have fully active glycolytic pathways and increased expression of AcPase (7). AcPase is speculated to regulate glycolysis by hydrolyzing 1, 3bisphosphoglycerate releasing Pi, which maintains ADP levels (3). In mammalian muscle cell lines AcPase enhances both glycolytic flux and fermentation rates (6). AcPase expression is enhanced by thyroid hormone levels in human cell lines suggesting that AcPase potentially plays a role in metabolism, homeostasis regulation, or a combination of the two (3). Overexpression of AcPase is observed during both cell differentiation and apoptosis suggesting that AcPase functions with the cell cycle, and the high expression levels suggest a role in tumour genesis (35). These results and the commonality of the carboxy-phosphate bond suggests that AcPase possesses a role in glycolysis regulation. 18 The N-terminus of a larger enzyme, E. coli HypF (hydrogenase maturation factor F), shares structural homology with AcPase suggesting that AcPase could function in conjunction with a protein complex. In the N-terminus domain of HypF, the AcPase-like domain acts to produce the necessary cofactors for the enzymatic function of hydrogenase. Therefore, in a potential complex AcPase could function similarly, in that it produces molecules that are necessary for enzymatic activity of the complex or producing cofactors necessary for function of another enzyme. AcPase is also speculated to function as a member of a protein complex. AcPase could facilitate a single step in the production of the final product produced by the protein complex. The complex potentially functions to transform a molecule into a different molecule where the hydrolysis of a carboxy-phosphate bond is one necessary step in that transformation. The E. coli HypF-N has been shown to aggregate faster and form amyloid fibrils faster than human muscle type AcPase which makes it the protein of choice for investigating this process. The difference between the two proteins is speculated to arise from the intrinsic differences between the two peptide sequences rather than the conformational changes in native state stability (37). Amyloid fibrils are formed from a variety of conditions that promote unfolding when starting with HypF-N (39). The HypF-N derived prefibrillar aggregates resemble the amyloid disease protofibrils and retain the cytotoxic nature of these aggregation products making this peptide optimal in studying aggregation and its associated diseases (38). This suggests that AcPase could potentially play a role in aggregation associated diseases. Introduction of Kinetics Kinetic investigations give insight into the function of enzymes by answering questions about optimum substrates and aiding in characterizing the enzymes function. Kinetic investigations give a quantitative outlook on enzyme activity. Aside from indicating optimum substrates, kinetic investigations can also indicate favourable reaction conditions, required cofactors, if an enzyme functions in both the forward and reverse direction, identify molecules that influence enzyme activity and specifics of enzyme- substrate interactions. Kinetic investigations use the relationship between substrate concentration and the conversion rates of substrate to product to characterize enzymatic activity. From this relationship, both Vmax and Km can be extrapolated, and these two parameters are used to describe 19 enzyme activity. Maximum velocity (Vmax) is the maximum rate of reaction; this is directly impacted by enzyme concentration and is a physical characteristic of the reaction. The Michaelis-Menten constant (Km) is a rate constant that describes enzyme and product dissociation and is intrinsic to the enzymatic reaction (38). When used as a dissociation constant Km describes the affinity an enzyme has for a substrate (38). There is an inverse relationship between Km and the enzyme-substrate interactions. A low Km indicates that there are strong interactions between the enzyme and substrate and high Km indicates that there are weak interactions. Vmax is a relative parameter that is directly influenced by enzyme concentration. Therefore, it cannot be used for the direct comparison of enzymatic activity with differing substrates. The comparison of overall effectiveness and efficiency of an enzyme is through the catalytic constant. The catalytic constant (kcat) is an intrinsic characteristic of an enzymatic reaction. kcat values are used to compare enzyme activity among differing substrates to determine the optimum substrate for a specific enzyme. In the case of Michaelis enzymes, kcat is also the turnover number and represents the rate constant k2 which is the slowest step of the reaction. Inhibition kinetic studies investigate the negative impacts a molecule possesses on enzyme activity. Inhibitors have differing negative effects on activity which depend on how they interact with the enzyme. Competitive inhibitors compete with the substrate for the active site and the interaction with the enzyme does not lead to any products. Competitive inhibitors occupy the active site and have no impact on Vmax, but influence how long it takes to reach Vmax, by decreasing the Km (39). Inhibitors that interact with the enzyme at a different location than the active site allow for the enzyme to associate with the substrate (making it an uncompetitive inhibitor) decreases Vmax and has no impact on Km (39). The last classification of inhibition, mixed inhibition is when the inhibitor possesses a combination of competitive and uncompetitive interactions. The inhibitor either binds to free enzyme or binds to enzyme bound to the substrate and decreases both Vmax and Km (39). Previous Kinetic Studies AcPase kinetics have been previously investigated by Paoli et al (1997), in a study that encompassed a broad range of kinetic assays including the comparison of a collection of substrates, a series of continuous assays, an inhibition study, and an investigation into the 20 potential reaction schematic of AcPase (3). In this study, AcPase was isolated and purified from bovine muscle and all kinetic assays were conducted in a pH 5.3, 0.1 M sodium acetate buffer. The first kinetic investigation was a series of steady state kinetic assays using acyl phosphates of varying carbon chain lengths, specifically 2, 3, and four carbon lengths and phenyl-acyl phosphate (3). The acyl phosphates were found to possess similar Km values (0.20, 0.30 and 0.15 mM respectively), while the kcat (181, 117 and 77 s-1, respectively) values varied over a larger range than the Km values (6). It was suggested this was due to the strong effect that the acyl phosphate pKa values (4.75, 4.87 and 4.81 respectively) had on hydrolysis (3). The wide range of substrates tested having similar Km values showed each substrate interacted with AcPase in the same manner, with the phosphate group. The differing kcat values showed that the hydrolysis rate was affected by something that did not affect binding which was the differing pKa values of the leaving group. These results reinforced that the slow step of the reaction was the release of the leaving group. The second kinetic investigation was a continuous kinetic assay of AcPase with benzoyl phosphate (BzP), 2- methoxy-benzoyl phosphate (2mBzP) and p-nitrobenzoyl phosphate (3). It was noted that out of this reaction series the p-nitrobenzoyl phosphate reaction progressed slower than the others which suggested that the nitrogen group had a negative impact on the hydrolysis reaction due to the difficulty of releasing the p-nitrobenzoate from the AcPase active site without the presence of an acidic group. The third investigation was an inhibitory study of phosphate and benzoate on the hydrolysis of benzoyl phosphate (3). Of the two potential inhibitors, phosphate had an inhibitory effect, decreasing activity. This was observed with a concentration of 5 mM and compared to the activity in the presence of 5 mM benzoate showed that benzoate had no inhibitory impact on activity. Further investigation into the inhibition of phosphate revealed that phosphate acted as a competitive inhibitor. The final study conducted was an investigation of a potential reaction schematic for the hydrolysis reaction of BzP by AcPase (6). The hydrolysis occurred in the presence of heavy water to reveal a potential reaction schematic. The presence of 18O in the phosphate group released and not in the benzoate showed that the water molecules attacked the phosphate group during hydrolysis (6). 21 Hydrogenase Maturation Factor, HypF The small size of acyl phosphatase makes it a good candidate to function as a domain of a larger protein. This is true in the case of the E. coli hydrogenase maturation factor, HypF (41). The hydrogenase maturation factor genes (hyp genes) code for the enzymes that function to produce the nickel coordinated hydrogenase active site (41). All six of the hyp genes are found in the same operon. HypF is one of the six maturation factors required to produce active hydrogenase. It is comprised of 783 amino acids and found in the N-terminus is an AcPase- like domain. This AcPase-like domain is speculated to facilitate the formation of both carbon monoxide (CO) and cyanide (CN-) from carbamoyl phosphate, as seen in figure 2.5 (4). The mechanism is unknown, but the first step of this conversion is speculated to hydrolyze the phosphate group, and the conversion of both products occurs from two carbamoyl phosphate molecules. The first is oxidized while the other is reduced producing both products at the same time. Both products are coordinated by the hypF N-terminus AcPase-like domain into the hydrogenase metal cluster. The two products are speculated to stabilize the active site metal ions maintaining their low oxidation states (4). The proposed mechanism for this is that Arg23 and Asn41 supports the substrate orientation for nucleophilic attack of a water molecule on the phosphate group. A comparison of both for AcPase and HypF- N revealed that the two enzymes follow similar mechanisms. Figure 2.5: The general chemical reaction that the N-terminus of HypF facilitates, the conversion of carbamoyl phosphate into cyanide- and carbon monoxide. 22 Chapter 3 Materials and Methods Introduction The materials and methods used in this study are compiled here in a comprehensive materials and methods section. The materials and methods for Chapter 4 Kinetic Investigations are found from “Preparation of the 1167pET21b Vector" to "AcPase Kinetic Assays." All primer sequences used in this study are found in Table. 3.1. Preparation of the 1167pET21b Vector Ligation The rrnAC1167 gene (Har. Marismortui AcPase gene) was amplified by colony PCR (cPCR). Each reaction had 1X GoTaq Master Mix (Promega), and 0.4 µM of each primer: 1167ecoRIF and1167xhoIR in a total volume of 25 µl (Table 3.1.) Each colony that was selected from a growth plate containing 1167 in pUC19 and was resuspended in the reaction volume with a pipette tip. The PCR reaction occurred in a Bio- Rad DNA Engine Dyad Peltier Thermal Cycler. The PCR program that was used began with an initial incubation step for one minute at 95 °C, followed by 30 cycles of the following: a 30- second incubation at 95 °C, followed by a 30 second incubation at 42 °C and ending with a 1:30- minute incubation at 72 °C. The final step in the PCR program was an incubation at 4 °C until the reactions were collected from the Thermal Cycler. 23 Table 3.1 The sequences and melting points of all primers used for sequencing and cPCR amplification in this study. Sequence Melting point (C) 1167ecoRIF GGAATTCGATGGCTCACACGCGAGCAC 58.7 1167xhoIR GGAGCTCCCAGCGGACTTCGAAGCC 65.2 T7 forward TAATACGACTCACTATAGGG 47 T7 term TATGCTAGTTATTGCTAG 47 Primer Both the empty pET21b vector and the cPCR amplified 1167 gene insert were doubledigested in preparation for the subsequent ligation. The double digests each had a reaction volume of 50 µl and included in each reaction was 1X Cutsmart buffer, 20 U of XhoI, 20 U of EcoRI and 1000 ng of DNA product. Each reaction was incubated for an hour in a 37 °C water bath. Both digested pET21b and digested 1167 gene insert were run on a 0.5% agarose gel with 1X TAE running buffer for approximately an hour at 90 V for gel purification. Gel purification procedures were as per the Omega Bio-Tek E.Z.N.A gel extraction kit (43). The ligation of the 1167 gene insert into the pET21b vector followed standard ligation procedure. The ligation reaction had a total volume of 20 µl and included in the reaction was 1X ligase buffer, 400 U of T7 ligase, 0.5 mM ATP and 100 ng of digested pET21b with a 3:1 molar excess of digested 1167 insert. The ligation reaction was incubated at room temperature overnight. Transformation The 1167pET21b ligation product was transformed into the E. coli competent strain DH5a. A stock of the DH5a cells, stored at -80 °C in 10% glycerol and LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1 mM NaOH), was thawed on ice. 50 µl of the DH5a competent cell stock was incubated with half the ligation reaction on ice for 30 minutes. This was followed by a 24 one- minute heat shock in a 42 °C water bath. Immediately after the heat shock, the cells were incubated on ice for two minutes before being transferred to a 1 mL culture of LB at 37 °C. The broth was incubated in a shaker (300rpm) at 37 °C for a 30- minute recovery step. The recovery culture was divided up among four different 20% agar LB plates containing 50 µg/ml ampicillin. The plates were incubated at 37 °C overnight. Verification of 1167pET21b Sequence Verification of the 1167pET21b ligation product sequence was accomplished by colony PCR (cPCR), restriction enzyme digestion and lastly DNA sequencing. The following cPCR proceeded as previously stated using the T7 forward and 1167xhoIR primers. Colonies that had positive results for the presence of the 1167 gene were grown in a 20 ml culture overnight. The entire culture was harvested, lysed and the plasmid was purified with the Omega Bio-Tek E.Z.N.A plasmid purification kit (44) with the following modification to the elution step: the elution buffer was brought to 65 °C in a heat block and the elution volume used was 30 µl. The elution step was completed twice, running the first elution volume over the column a second time to increase plasmid yields. Purified 1167pET21b was sequenced at the UNBC Genetic Facility using both T7 forward and T7 terminator primers. The sequencing results are found in the appendix figures A2 and A3. Sequences were analyzed and assembled using FinchTV (Geospiza). AcPase Expression and Purification Expression The transformation procedure was carried out as previously stated with the following modifications: the competent cells used were the Rosetta 2 (pLysS) E. coli expression strain and the 20% agar LB plate contained both 50 µg/ml ampicillin and 34 µg/ml chloramphenicol. After the purified 1167pET21b vector was transformed into Rosetta 2 (pLysS) competent cells, a colony was used to inoculate a 20 ml LB culture containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol that was incubated overnight in a shaker (300rpm) at 37 °C. The overnight culture was used to inoculate a one litre culture with a 1/100 ratio that was incubated in a shaker 25 (300rpm) at 37 °C. When the optical density (OD) of the culture ranged between 0.6- 0.8 1% (w/v) lactose was added. Four hours after the lactose induction the culture was transferred to a one litre centrifuge tube and harvested by centrifugation for 20 minutes at 4 °C in an Avanti HP20XPI floor centrifuge using a JLA 8.1 rotor at 3000 g. The cell pellet was resuspended in a smaller volume of LB media and transferred to a pre- weighed 50 ml conical tube for a 20minute centrifugation step at 3000 g and 4 °C in an Allegra X12R table top bucket centrifuge. The pellet was weighed and resuspended in 10 ml of lysis buffer (0.2 mM NaCl, 1 mM EDTA, 1 mM b-mercaptoethanol 5% glycerol pH 7.4) and stored at -20 °C overnight. Lysis The frozen cell suspension was thawed on ice, followed by the addition of 10- 15 ml of lysis buffer per 4 g of cells harvested. After the addition of lysis buffer lysozyme was added to a final concentration of 1 mg/ml and allowed to incubate for a minimum of half an hour at room temperature. Depending on the viscosity of cell lysate, more lysis buffer was added before the cell lysate was sonicated (Fischer Scientific Sonic Dismembrator, model 100). Sonication was repeated four- six times at power level ten, following the pattern of 30 seconds on and oneminute rest. The cell lysate was transferred to a 50 ml round bottom centrifuge tube for centrifugation at 25000 g for 20 minutes at 4 °C in an Avanti HP- 20XPI floor centrifuge using a JA 25.50 rotor. The supernatant was then transferred to a new 50 ml round bottom centrifuge tube, and 1% (w/v) streptomycin was added followed immediately by inverting the tube and a second centrifugation step as previously described to precipitate genomic DNA. The resulting supernatant was filtered through a 0.2 µm syringe filter in preparation for purification of the AcPase protein. AcPase Purification The filtered cell lysate was purified with a GE Healthcare fast flow His- trap column (5 ml column volume (CV)) on an AKTA Purifier FPLC with a flow rate of 1 ml/min. The buffers used were: Buffer A (50 mM NaH2PO4, 300 mM NaCl, 5 mM imidazole, pH 8.0), Buffer B (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole, pH 8.0), 20% ethanol and ddH2O. All buffers were filtered with a 0.2 µm nylon membrane filter (Millipore) and degassed for a minimum of 26 30- minutes before use. The column was first equilibrated with five CV of 20% ethanol, followed by a five CV equilibration in ddH20 and ending with a final five CV equilibration in Buffer A. The entire volume of the filtered cell lysate was injected onto the column using a super loop (GE Healthcare), followed by a 12 CV wash with Buffer A. Following the wash, Buffer B was introduced over a ten CV gradient ranging from 0% Buffer B to 100% Buffer B. Collection of one mililitre fractions began at the start of the Buffer B gradient. 100% Buffer B was held for five CV before the column was re-equilibrated in Buffer A for five CV. Fractions were taken from different points of the purification and assessed on a Coomassie blue stained 15% SDS-PAGE. The SDS-PAGE samples were prepared by the addition of 1X loading dye (2% SDS, 0.1% bromophenol blue, 10% glycerol, 1.2% b mercaptoethanol, 50 mM Tris, pH 6.8) followed by boiling for five minutes. Fifteen microlitres of each sample was loaded and the SDS gel was run for 45- minutes to an hour at 20 mA in 1X SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). The gel was stained for an hour in Coomassie blue dye (2.92 mM Coomassie blue R250, 45% ddH2O, 45% ethanol, 10% glacial acetic acid) on a shaker followed by an overnight incubation in destain (10% glacial acetic acid, 50% ddH2O, 40% ethanol). Identification of Purified AcPase Protein Size exclusion chromatography was performed using a Superdex 200 sizing column (GE Healthcare) on an AKTA Purifier FPLC after His- trap purification to verify the molecular weight of the purified protein. The column was equilibrated first in 1.5 CV of water, followed by another 1.5 CV of desalting buffer (0.5 M NaCl, 50 mM Tris). The flow rate used for the size exclusion purification was 0.5 ml/min. All peaks detected at 280 nm were subsequently run on an SDS-PAGE as previously described with the amendment that the gel was silver stained using the Bio-Rad Silver Stain Plus kit (44) to identify in which fraction is the Har. marismortui AcPase protein. All fractions that potentially contained Har. marismortui AcPase protein were tested for functional activity, concentrated, desalted and re-assessed on a 15% SDS- PAGE before a final concentration was determined by Nanodrop (ND-1000 spectrophotometer). Each activity test had a total volume of 350 µl including reaction buffer (20 mM piperazine, 0.4 M KCl, 1 mM EDTA, pH 5.3), with 2mM BzP (benzoyl phosphate) and 15 µl of each enzyme fraction. Each fraction 27 activity test was then consolidated, concentrated and desalted using a vivaspin500 desalting column with a 3 kDa molecular weight cut- off (GE Healthcare) using the desalting buffer. Substrate Synthesis Micro-Synthesis Revised methods for the synthesis of 2mBzP (2- methoxy-benzoyl phosphate) (7) were used for the synthesis of both 2mBzP and 4mBzP (4- methoxy-benzoyl phosphate) with the following amendments. The reaction conditions used for each substrate were 25:1(v/v) of THF ((tetrahydrofuran): TEA (triethylamine), in a final volume of 520 µl prepared on ice. The methoxy-benzoyl chloride(s) were added (0.147 mmol 2mBzP and 0.146 mmol 4mBzP) at room temperature followed by the addition of pyridine to a final volume of one mililitre. The mixture was thoroughly mixed before the addition of 85% (v/v) H3PO4 to a final volume of 1.1 ml. The reaction proceeded overnight at room temperature and products identified by HPLC (Waters) with a Waters C18 reverse phase column (with a flow rate of 1 ml/min, a running pressure of 1500 psi, a six- minute run time and a 10 µl injection volume), using a 50% acetonitrile solvent. Macro-Synthesis The methods of the micro-synthesis of each methoxy substrate were revised (7) for the macro synthesis. The general synthetic mechanism is illustrated in figure 3.1. The reaction conditions were made on ice with 16.8:1(v/v) of THF: TEA, to a final volume of 31.8 ml. The methoxy-benzoyl chloride(s) were added (13.3 mmol 2mBzCl and 13.3 mmol 4mBzCl) at room temperature followed by the additions of 18% v/v of pyrimidine to THF (5.4 ml). After being mixed thoroughly with the reaction mixture, 1.41 moles of H3PO4 was added and the reaction proceeded overnight at room temperature with constant stirring. The product was precipitated out of solution with the addition of ten volumes of cold acetone and 19.95 mmol LiCl2. The initial purification steps involved several recrystallization steps that included dissolving the product in one volume of water (heating in a hot water bath as needed), adding ten volumes of cold acetone and filtering the precipitated product by gravity filtration. 28 Figure 3.1: Top: The condensed mechanism of the synthetic approached taken to synthesize 2- methoxy-benzoyl phosphate. Bottom: The condensed mechanism of the synthetic approach used in the synthesize 4- methoxy-benzoyl phosphate. Verification of Substrates The verification of substrates and determination of inorganic phosphate contamination were accomplished by a wavescan and conducting a malachite green assay provided for the R&D malachite green phosphate detection kit (45). The wavescans ranged from 200 nm to 800 nm and were made using a DU 800 Spectrophotometer (Beckman Coulter) with a one centimeter path length quartz cuvette. The following three dilutions of a stock solution of each substrate in the reaction buffer (20 mM piperazine, 0.4 M KCl, 1 mM EDTA, pH 5.3) were made:1/5, 1/10, and 1/100. The malachite green assay was carried out following the protocols the R&D malachite green phosphate detection kit for microplates (45). A Synergy 2 microplate reader (Biotek) was used for detection. Purification Each substrate was purified using a 5RPColigo HPLC column (Amersham Biosciences), with a column volume (CV) of 2.5 ml, on an AKTA Purifier FPLC. The following buffers were used: Buffer A (10 mM acetic acid, 2% acetonitrile), Buffer B (10 mM acetic acid in methanol, 2% acetonitrile), and an 80% acetonitrile solution. All buffers used were filtered and degassed as previously stated. The column was first equilibrated with 80% acetonitrile for five CV, followed 29 by a five CV equilibration with Buffer B and ending with a five CV equilibration in Buffer A. One hundred microlitres of stock substrate solution (substrate dissolved in Buffer A) was injected onto column, and the collection of 1.5 ml fractions was initiated. The injection was followed by a two CV Buffer A wash. After the wash, 50% Buffer B was held for two CV before a gradient ranging from 50% Buffer B to100% Buffer B over ten CV was initiated. One hundred percent of Buffer B was held for five CV and fractions were collected until the first CV of 100% Buffer B had completed. Concentration was determined by performing a 1/100 dilution of the stock and adding the same volume of 10 M NaOH and the extinction coefficient (BzP ∆E 0.63 A/cm-1mM-1 at 283 nm, 2mBzP ∆E 0.1997 A/cm-1mM-1 at 278 nm and 4mBzP ∆E 0.3399 A/cm1 mM-1 at 258 nm) was used to determine the concentration. AcPase Kinetic Assays All kinetic reactions occurred in the reaction buffer (20 mM piperazine, 0.4 M KCl and 1 mM EDTA, pH 5.3) unless otherwise stated. The wavelengths used for monitoring each kinetic reaction were as follows: BzP at 283 nm, 2mBzP at 278 nm and 4mBzP at 258 nm. Each reaction was set up with a total volume of 350 µl, where 150 µl was made up of stock substrate dilution and 200 µl of AcPase enzyme reaction stock. The enzyme stocks were stored on ice and the reactions occurred at room temperature. BzP, 2mBzP, and 4mBzP A 20 mM stock of each substrate was used to set up the dilutions based on the Km of each reaction following the general pattern of: ¼ Km, ½ Km, Km, 2 Km, and 3 Km. The final concentration of AcPase used in each assay was 2.64x10-4 mM for BzP and 1.83x10-4 mM for both 2mBzP and 4mBzP. All assays occurred in a 1 mm path length quartz cuvette and were monitored over a two- minute period in which the absorbance was measured every six seconds in a DU 800 Spectrophotometer (Beckman Coulter). Each assay was performed in triplicate. Each reaction slope was determined using Excel and plotted using the Michaelis-Menten formula in Kalidagraph (Synergy Software). 30 Reversible Reaction The substrates used in testing the reverse chemical reaction were benzoic acid and phosphate, which was monitored at 283 nm for the formation of BzP. The stocks used were 500 mM sodium phosphate, pH 5.3 and 20 mM benzoic acid. The concentration of benzoic acid was held constant at 5 mM, and inorganic phosphate concentrations varied as follows: 5, 30, 50 and 100 mM. The final concentration of AcPase used in each assay was 1.83x10-4 mM. All assays occurred in a 1 mm path length quartz cuvette and were monitored over a two- minute period in which the absorbance was measured every six seconds in a DU 800 Spectrophotometer (Beckman Coulter). The data was plotted in Excel. Acetate Inhibition The substrate used to test for the inhibitory effects of acetate was benzoyl phosphate, monitored at 283 nm. The stocks used were 2 M sodium acetate, pH 5.3 and 5 mM benzoic acid. The concentrations of BzP that were used are as follows: 0.375, 0.75, 1.5, 3 and 4.6 mM. The concentrations of acetate used were: 0, 50, 100, 200 and 400 mM. The final concentration of AcPase used in each assay was 1.83x10-4 mM. All assays occurred in a 1 mm path length quartz cuvette and were monitored over a two- minute period in which the absorbance was measured every six seconds in a DU 800 Spectrophotometer (Beckman Coulter). Each assay was done in triplicate, and the averages of each velocity were plotted using the Lineweaver-Burk manipulation of the Michaelis-Menten equation. The data was fit to the Lineweaver-Burk equation in Kalidagraph (Synergy Software) and plotted in Excel. Carbamoyl phosphate Activity The first test of AcPase activity with carbamoyl phosphate used the hydroxamate assay as a detection method following the protocols by Fowler et al (2011) with the following exceptions (46): All reactions were heat inactivated for one minute at 100 °C, and all reagents used were made fresh. Two sets of assays were conducted a set with acetyl phosphate as a positive control, and a set with carbamoyl phosphate. The stocks used were 100 mM carbamoyl phosphate and 20 mM acetyl phosphate. Each reaction had a total volume of 300 µl with a working concentration of carbamoyl phosphate (or acetyl phosphate) of 3 mM and using 150 µl of a stock enzyme 31 solution with a final concentration of AcPase of 1.83x10-4 mM. This assay had two time frames: two minutes and overnight. The second test of AcPase activity with carbamoyl phosphate used the malachite green assay was carried out following the protocols provided with the R&D malachite green phosphate detection kit for microplates (45). Each reaction had a total volume of 300 µl with the following working concentrations of carbamoyl phosphate: 0, 0.1, 0.75, 1.5, 3, 6 and 10 mM. The final working concentration of AcPase was 1.83x10-4 mM. A set of blanks were also made containing the same final concentration of carbamoyl phosphate and final volume while lacking AcPase. The blank was used as a correction for the Pi concentrations detected by the malachite green assay to assess the activity of AcPase. A Synergy 2 microplate reader (Biotek) was used for detection of inorganic phosphate. The data was plotted and fit to the Hill equation in Kalidagraph (Synergy Software). 32 Chapter 4 Kinetic Investigation Introduction The comparison of enzymatic activity with differing substrates gives insight into both specificity and mechanism. These kinetic investigations determine molecular characteristics of potential substrates, influences on enzyme- substrate interactions, promoters of faster enzymatic activity and the overall efficiency of the enzyme. Comparing enzyme activity with different substrates can elucidate parts of the enzymatic mechanism, the catalytic residues of the active site and lastly, determines activity changes between each substrate. Kinetic studies give a quantitative outlook on enzymatic activity, revealing optimum substrates, favourable reaction conditions and potential required cofactors for the reaction. Additionally, the reversibility of enzymatic reactions can be studied and provides further insights into the molecules that impact enzymatic activity as well as an enzyme’s affinity for a given substrate. The relationship between activity and substrate concentration of a given enzyme concentration allows the parameter Km to be determined. The Km of an enzymatic reaction approximates the dissociation constant and is thus a constant for E and S formation from the dissociation of the ES complex, whereas the Vmax describes the maximum velocity of the reaction at the enzyme concentration used in that reaction (38). These two parameters are relative to each kinetic assay, as they are influenced by both the enzyme concentration and the substrate in question. A second kinetic parameter can be calculated to further describe an enzymatic reaction: the kcat of an enzymatic reaction describes the efficiency of an enzyme in respect to the substrate in question (38). This parameter is used to directly compare differing enzymes or compare differing substrates for a given enzyme. From kcat and Km the specificity constant of an enzyme can be described providing a different means to compare different substrates with a given enzyme (38). The two substrates synthesized and compared in this study were 2- methoxy-benzoyl phosphate (2mBzP) and 4- methoxy-benzoyl phosphate (4mBzP); the molecular structures of both are presented in figure 4.1. These two molecules differ only in the position of the methoxy group which is the electron-withdrawing group they both possess. The different locations of the electron-withdrawing group were expected to have different impacts on both the carboxy 33 phosphate bond and the AcPase facilitated hydrolysis of the carboxy phosphate bond. The comparison of these two substrates was therefore expected to suggest a favourable position of the methoxy group for Har. marismortui AcPase activity allowing for the partial characterization of the native substrate. Figure 4.1 The structures of 2- methoxy- benzoyl phosphate (2mBzP) and 4- methoxy- benzoyl phosphate (4mBzP), both synthesized and purified for kinetic studies with Har. marismortui AcPase. The kinetic study that originally investigated AcPase activity with 2mBzP isolated three different bovine AcPase isoforms (8). Two isozymes were organ-specific common type isozymes and the third was a muscle type isozyme was isolated from bovine muscle (8). This study conducted by Paoli et al (1995) investigated AcPase activity with all three isozymes with both substrates BzP and 2mBzP. The two organ-specific AcPase common type isozyme activity assays with both substrates 2mBzP and BzP produced similar Km values. The Vmax values produced from both organ-specific isozymes revealed differences in enzymatic activity with these substrates. The Vmax for 2mBzP activity was on average 10% lower than that of BzP activity (8). The muscle isozyme revealed that activity with 2mBzP and BzP had similar Km values, whereas the Vmax associated with 2mBzP activity was 30% lower than that of BzP (8). The results from both the AcPase organ-specific isozymes and the AcPase muscle type isozyme indicated that 2mBzP and BzP share similar properties suggesting that 2mBzP is another molecule that can be used for AcPase characterization. AcPase characterization with 4mBzP has not been investigated previously. The materials and methods used in this Har. marismortui AcPase kinetic investigation are compiled in Chapter 3. 34 Acyl phosphatase Cloning, Expression, and Purification Har. marismortui AcPase was cloned, expressed and purified for use in kinetic studies for the kinetic characterization of this enzyme with various substrates. The Har. marismortui AcPase gene was amplified from DH5a E. coli containing the gene in pUC19 by colony PCR, a variation on the PCR procedure in which ligation colonies are tested for the presence of the desired gene and plasmid. The template in this PCR procedure arises from the individual cells added to the PCR mix when the colony is selected by a pipette tip and ‘swished’ in the mixture. The amplified gene was purified for the subsequent ligation into the expression vector pET21b. The ligation success was confirmed by a second colony PCR reaction detecting the presence of the 1167 gene in the pET21b vector using the vector specific forward primer T7 forward and the 1167 gene specific reverse primer 1167xhoRI (figure 4.2). Each colony PCR reaction that was run on an 0.5% agarose gel showed the success of the ligation reaction. The agarose gel shows if the 1167 gene was amplified and is present in the pET21b vector, or if the 1167 gene was not amplified and not present in pET21b (figure 4.2). 1 2 3 4 5 Bp 700 500 400 300 200 150 100 75 50 25 Figure 4.2 The 0.5% agarose gel visualizing colony PCR DNA to assess the presence of 1167 gene in the pET21b vector. Lane: 1) ladder, 2) colony 1, 3) colony 2, 4) colony 3 and 5) negative control The 1167pET21b vector was tested for the presence of the Har. marismortui AcPase gene in the vector (pET21b) by colony PCR. In each of the three random colonies that was tested, a band was present between the 400 bp and 300 bp markers from the ladder (well 1) whereas the negative colony PCR reaction (well 5) had no band within this size range. These bands that were approximately 350 bp in length and correspond to the sequence length expected 35 between the forward primer site (T7) and the reverse primer site (1167XhoRI) and positively confirmed the presence of the 1167 gene in the pET21b vector. Of the three colonies tested (colony 1, 2 and 3), colony 2 was sequenced at the UNBC Genetics Facility using both the T7 promoter and T7 terminator primer; both the forward and reverse primer sequences are available in the appendix (A2 and A3 respectively). The forward and reverse reaction sequences were subsequently aligned with the original gene sequence and revealed no DNA mutations. The 1167pET21b vector was transformed into the E. coli expression strain Rosetta 2 (pLysS) and two colonies were selected from the overnight transformation plate and used to inoculate two individual overnight cultures that were then used to inoculate two independent, larger, one litre expression cultures. Each lactose-induced expression culture was harvested and lysed and Har. marismortui AcPase was purified independently of one another. Both expression culture cell lysates were purified by FPLC using a His-trap column with the same purification protocol. The imidazole rings of histidine residues in the 6X His-tag interacted with nickel ions coordinated in the His-trap column. The Har. marismortui AcPase C- terminus 6X His tag formed stronger interactions with the column than the single histidine residues in other protein sequences. The additional imidazole present in the elution buffer (Buffer B) used during purification caused the bound proteins to be competed and displaced by the imidazole, allowing the bound proteins to elute from the column. The FPLC chromatogram depicting the purification of Har. marismortui AcPase (figure 4.3) indicated that there were two main elution peaks that absorbed at 280 nm indicating each contained protein. Each peak on the chromatogram contained protein that did not interact with the column, interacted weakly with the column or interacted strongly with the column. The first peak, the wash peak, ranged from approximately 5 ml (one column volume) after injection to approximately 55 ml. The wash peak was broad and high in both 280 nm and 254 nm representing the cellular proteins and nucleic acids that did not interact with column. The increased the imidazole concentration in the elution buffer (Buffer B) caused the bound proteins to the column to elute, producing two individual elution peaks. 36 UNICORN 5.11 (Build 407) Result file: C:\...\default\NiNTA\AJGNiNTA018 AJGNiNTA018:10_UV1_280nm AJGNiNTA018:10_Cond AJGNiNTA018:10_Inject AJGNiNTA018:10_UV2_254nm AJGNiNTA018:10_Conc AJGNiNTA018:10_Fractions AJGNiNTA018:10_Logbook mAU 5000 4000 3000 2000 1000 0 1 3 5 7 9 12 16 20 24 28 32 36 40 44 48 52 0 20 40 60 80 100 Waste 120 140 ml Figure 4.3 The FPLC chromatogram of the Har. marismortui AcPase purification using a His-trap column. Legend: The blue line indicates UV absorption at 280 nm, the red line indicates UV absorption at 254 nm, the green line indicates %Buffer B, the brown line indicates conductance, the orange dashed lines indicate the fractions collected, and the pink line indicates the injection of sample. The Y axis measures absorption in mAU and the X axis indicates run volume in ml. The chromatogram was generated in Unicorn 5.11 After the increase in imidazole concentration, two individual elution peaks were observed on the AcPase purification FPLC chromatogram. The first elution peak (fraction 9- 15) was 37 considerably higher and eluted earlier than the second elution peak (fraction 16- 22). The first elution peak contained peptides that had weaker interactions with the column, as a smaller imidazole concentration was required to displace these bound proteins. The second peak required a larger imidazole concentration to displace the bound proteins indicating that these proteins formed stronger interactions with the column. The composition both elution peaks was assessed to confirm which elution peak contained the 6X His-tagged Har. marismortui AcPase. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a common method used to visualize and identify protein samples based on molecular weight. This technique was used to identify which of the two elution peaks, from the previous FPLC purification, contained Har. marismortui AcPase. Both expression cultures were assessed on the same SDSPAGE (figure 4.4). For each expression, the fractions that were loaded on the gel were: cell lysate, fractions from the peak boundaries, and fractions from the middle of each peak. 1 2 3 4 5 6 7 8 9 10 11 12 kDa 66 45 36 29 24 13 20k 14 6.5 Figure 4.4 The coomassie blue stained 15% SDS-PAGE gel visualizing the FPLC purification of Har, marismortui AcPase. The wells were loaded from left to right: 1) cell lysate 1, 2) fraction 111,3) fraction 12-1, 4) fraction 16-1, 5) fraction 19-1, 6) fraction 22-1, 7) cell lysate -2, 8) fraction 11-2, 9) fraction 12-2, 10) fraction 16-2, 11) fraction 19-2, 12) fraction 22-1-2 and13) low molecular weight marker. The SDS-PAGE visualizing the FPLC purification of Har. marismortui AcPase compares the initial cell lysate (before purification) to the fractions taken during the FPLC purification to identify which fraction(s) contained the 6X His-tagged Har. marismortui AcPase. The cell lysate fractions each contained cellular proteins and nucleic acids. The cell lysate showed the relative concentrations of cellular proteins present, including Har. marismortui AcPase before 38 purification. In each cell lysate fraction, there was a relatively intense band, with a molecular weight that was approximately 10 kDa that corresponded to the known molecular weight of Har. marismortui AcPase. The presence of these intense bands in each cell lysate indicated that Har. marismortui AcPase was successfully expressed. The 10 kDa AcPase band observed in the cell lysate was absent in fractions from both the first and the second elution peaks (figure. 4.4). The first elution peak contained mainly larger proteins while the second elution peak contained smaller proteins. This trend was observed in both expression cultures. The second elution peak contained intense bands at approximately 20 kDa, double the expected molecular weight for Har. marismortui AcPase (figure. 4.4). The investigation of the these 20 kDa bands was expected to indicate if Har. marismortui AcPase was present and suggest that the observed doubling in molecular weight was caused by the SDS-PAGE conditions that were used. Gel filtration chromatography separates native proteins by size, whereas SDS-PAGE separates proteins under denaturing conditions. Gel filtration provided a different method for separating and identifying the proteins that were present in the second elution peak. Gel filtration was used to determine if the 20 kDa band identified by SDS-PAGE is either an artifact of electrophoresis or is present after gel filtration. The investigation of the protein composition of the intense 20 kDa band was expected to suggest the cause for the observed doubling of the molecular weight on the SDS-PAGE (figure 4.4). The entire second elution peak from the Histrap purification was loaded on the S200 sizing column. The chromatogram of the purification of the second elution peak from the S200 column is illustrated in figure 4.5. The proteins that were present on the S200 sizing chromatogram varied in size. Most of the proteins that were present were smaller proteins as most of the peaks on the S200 chromatogram eluted the column from fractions 26- 53 (figure 4.5) ranging from 35 kDa- 240 Da. The first peak (fractions 14- 16) was a short and broad peak that was composed of larger peptides (160 kDa). The third peak (fractions 26- 33) was the second largest peak overall on the S200 chromatogram and correlated to proteins with an approximate molecular weight of 10 kDa. The molecular weight of this peak suggested that this peak contained Har. marismortui AcPase, as there was no peak correlating to a protein with a molecular weight of 20 kDa. 39 UNICORN 5.11 (Build 407) Result file: C:\...\default\S200HS009 S200HS009:10_UV1_280nm S200HS009:10_Conc S200HS009:10_Logbook S200HS009:10_UV2_254nm S200HS009:10_Fractions S200HS009:10_Cond S200HS009:10_Inject mAU 200 150 100 50 0 1 2 3 4 5 6 7 8 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 0 50 100 150 ml Figure 4.5 The FPLC chromatogram of Har. marismortui AcPase sizing purification with a S200 sizing column. Legend: The blue line indicates UV absorption at 280 nm, the red line indicates UV absorption at 254 nm, the green line indicates %Buffer B, the brown line indicates conductance, the orange dashed lines indicate the fractions collected, and the pink line indicates the injection of sample. The Y axis measures absorption in mAU and the X axis indicates run volume in ml. The chromatogram was generated in Unicorn 5.11 40 SDS-PAGE was used to visualize the peaks from the S200 size exclusion separation of the His- trap second elution peak. The size exclusion peak that contained Har. marismortui AcPase can also be identified from the SDS-PAGE (figure 4.5). The SDS-PAGE was loaded with cell lysate (the starting material from the previous His-trap purification), and fractions from the various S200 size exclusion peaks. Due to the dilution of the protein concentration, the SDSPAGE was silver stained to visualize the proteins that were present. Figure 4.6 shows the silver stained gel visualizing the protein content of each sizing peak. kDa 66 45 36 29 24 1 2 3 4 5 6 7 8 20 14 6.5 Figure 4.6 The silver stained 15% SDS-PAGE gel visualizing the FPLC S200 size exclusion of Har. marismortui AcPase. The wells were loaded from left to right: 1) low molecular weight marker, 2) cell lysate, 3) fraction 23, 4) fraction 26, 5) fraction 27, 6) fraction 29, 7) fraction 31 and 8) fraction 32. The silver stained SDS-PAGE visualized the peaks of interest from the S200 purification of Har. marismortui AcPase. The peaks that were of interest were the second peak (~12.4 kDa) and the third peak (~10 kDa) on the S200 sizing chromatogram (figure 4.5). The molecular weight range between these two peaks suggested that they most likely contained Har. marismortui AcPase. The first three fractions of the third peak (~10 kDa) contained a band with an approximate molecular weight of 10 kDa on the SDS-PAGE gel. These three fractions (2628) contained a protein with the expected molecular weight of the Har. marismortui AcPase present in the cell lysate indicating that this protein is likely Har. marismortui AcPase. The three fractions (26- 33) that likely contained Har. marismortui AcPase on the FPLC S200 sizing chromatogram were pooled and purified by the His-trap column. The comparison between the two SDS-PAGE results (figure 4.4 and figure 4.6) gels from the His-trap purification and S200 sizing purification showed that Har. marismortui AcPase behaves differently on an SDS-PAGE in differing sample conditions. The preparation of each set of 41 samples was the same, they each were mixed with 1X SDS sample dye and boiled for a minimum of five minutes and the same volume of each sample was also loaded on the SDSPAGE. The difference that arises from the two sets of samples is the concentration of Har. marismortui AcPase. Har. marismortui AcPase possesses a lower pI and because of this, it is a naturally negative protein. At higher concentrations of this protein the natural high negative charge prevents the sample in acquiring a uniform coating of SDS detergent. This causes the protein to travel along the SDS-PAGE gel slower than expected resulting in observed molecular weight of the protein to be higher than the expected molecular weight. While in lower concentrations, Har. marismortui AcPase acquires a uniform coating of SDS detergent molecules in the sample dye and it travels along the SDS-PAGE as expected. This ‘quirk' of Har. marismortui AcPase behaving differently on SDS-PAGE gel depending on the concentration of AcPase is uncommon among proteins. Originally this phenomenon was thought to be due to age of the b-mercaptoethanol present in SDS sample dye. However, freshly prepared SDS loading dye was used to prepare a second Coomassie stained gel and revealed the same trend with an observed molecular weight that was double the expected for Har. marismortui AcPase. This trend is observed with other halophilic proteins as well. In higher concentrations, the observed molecular weight from an SDS-PAGE gel is larger than expected (47). Halophilic proteins have shared adaptations to remain both soluble and function and the trend of higher observed molecular weights by SDS-PAGE gel in high sample conditions is a consequence of these shared adaptations (47). Substrate Synthesis The original method for the synthesis of 2mBzP was revised from a prior synthetic method that was used in the synthesis of BzP for investigating AcPase activity (8). Simply, the synthesis was a two- step synthesis: the first step produces an anhydride and the second step cleaves this anhydride to yield the final product an unwanted carboxylic acid byproduct. The first step combines 2- methoxy-benzoic acid (2mBz acid) and 2- methoxy- benzoic chloride (2mBzCl) producing 2- methoxy-benzoic anhydride (7). The resulting anhydride then reacts with phosphoric acid, cleaving the anhydride into 2mBzP and 2mBz acid (8). The theoretical yield of 2mBzP from this method is 50% when both steps go to 100% completion. 42 This study investigated a revised method for the synthesis of 2mBzP and 4mBzP. The revised synthetic method removes the necessity to first produce an anhydride before producing the final product. The revised method reacts 2mBzCl directly with phosphoric acid to produce 2mBzP (figure 2.1). The revised method is a more direct synthesis and is theorized to produce more carboxy phosphate product and minimize the production of the carboxylic acid byproduct making it the superior method. This direct synthetic method was used to synthesize both 2mBzP and 4mBzP, which were subsequently used in the kinetic characterizations of Har. marismortui AcPase. Micro-Synthesis The micro-synthesis was used as an initial test of the viability of the revised synthetic method before a large-scale synthesis using this method was conducted. The micro-synthetic reaction allowed an initial comparison of the original synthetic and this revised synthetic method to be conducted determining which method was optimal in producing 2mBzP and 4mBzP. The viability of the revised synthetic method was assessed by reaction success, purity of the synthesized product, and lastly, comparing the product yield of the two synthetic methods. The first step in assessing the viability of the revised synthetic method was determining reaction success. HPLC with a C18 reverse phase column was used to determine if the reaction had occurred and if 2mBzP was synthesized. The HPLC was used to detect the presence of both 2mBzP and 4mBzP in their respective synthetic reaction. HPLC was also used to detect the presence of both 2- methoxy-benzoic acid and 4- methoxy-benzoic acid, respectively in the appropriate synthetic reaction which confirmed production of each respective byproduct of the revised synthetic method. The HPLC analysis on a Waters C18 column at 278 nm showed the presence of two unique molecular species present in the 2mBzP synthesis reaction. The presence of two molecular species suggested that both 2mBzP and 2mBz acid were produced in the revised synthesis reaction (figure 4.7). These peaks were identified by using the retention time of each peak. The comparison of the average retention times for 2mBz acid identified the second peak as 2mBz acid (figure.4.7). The first peak was then identified as 2mBzP based on the wavelength of detection and the shorter retention time of this peak. 43 MAu x10-2 (278 nm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Retention time (min) Figure 4.7 The HPLC data constructed into a visual representative spectrum illustrating the relative heights and retention times of each peak obtained from the analysis of 2mBzP synthesis product at 278 nm. The HPLC data for each synthesis reaction (2mBzP and 4mBzP) revealed that at 278 nm there were two unique molecular species in each synthetic product (Table 4.1). In each case, the methoxy-benzoic acid was produced alongside the desired methoxy-benzoyl phosphate product. The methoxy-benzoyl phosphate product in each case, had a shorter retention time than the methoxy-benzoic acid byproduct. At 278 nm 2mBzP and 2mBz acid have the greatest absorption differences. However, at this wavelength 4mBzP and 4- methoxy-benzoic acid (4mBz acid) do not have the greatest absorption differences. Table 4.1 The HPLC data at278 nm showing the retention time (RT) of each peak, the peak height of each peak and the identification of each peak for both the 4mBzP and 2mBzP synthesis product from the revised synthetic method. Substrate RT (min) Peak Height (AU) ID 1 190915 4mBzP 1.35 478205 4mBz acid 1.033 26500 2mBzP 1.367 42083 2mBz acid 4mBzP 2mBzP 44 The HPLC analysis at 258 nm proceeded as previously described for 278nm and revealed three individual peaks indicating that there were three unique molecular species present in each synthesis reaction (figure 4.8). The comparison of retention times between the two sets of HPLC data indicated that the first two peaks were 2mBzP and 2mzBz acid respectively. The final peak, however, was not present at 278 nm suggesting that this molecular species was initially not detected. The combination of the detecting wavelength, retention time and the synthesis method (the revised method does not produce an anhydride product) suggested that this peak was likely the pyridine component of the synthesis reaction system. MAu x10-2 (258 nm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Retention Time (min) Figure 4.8 The HPLC data constructed into a visual representative spectrum illustrating the relative heights and retention times of each peak obtained from the analysis of 2mBzP synthesis product at 258 nm. The HPLC data for both synthetic products, 2mBzP and 4mBzP, revealed that at 258 nm there was an unexpected third peak (Table 4.2). Two of these peaks were previously observed at 278 nm and were identified (Table 4.1). These first two peaks were identified as either the 2mBzP or 4mBzP synthetic reaction and their respective methoxy-benzoic acid byproduct. The third peak, not previously observed at 278 nm, represents a molecular species that was present in each synthetic product and was observed at 258 nm. The combination of the detection wavelength and retention time of this peak identified it as pyridine. In both synthetic products, leftover pyridine was present. 4mbzP and 4mBz acid have the greatest difference in absorption at 258 nm, this wavelength was therefore optimal in detection of 4mBzP. In both sets of HPLC data each synthesis produced both methoxy-benzoyl phosphate and the respective methoxy-benzoic acid. The first peak in each synthetic reaction was the methoxybenzoyl phosphate product and the second peak was the respective methoxy-benzoic acid 45 byproduct. The production of the respective methoxy- benzoic acids were unconfirmed byproducts of the revised synthesis method, but a confirmed byproduct in the original synthetic method. The revised reaction conditions limited the amount of water that was present in order to yield more methoxy-benzoyl phosphate product while decreasing the production of methoxybenzoic acid. The water present was introduced with the addition of phosphoric acid. The presence of the methoxy-benzoic acids in each respective synthetic reaction showed that the water limiting condition still produces the carboxylic acid byproducts and further purification steps are required. Table 4.2 The HPLC data at 258 nm showing the retention times (RT) of each peak, peak height and the identification of each peak for both 4mBzP and 2mBzP synthesis product from the revised synthetic method. Substrate Synthesis 4mBzP 2mBzP RT (min) Peak Height (AU) ID 1 335827 4mBzP 1.367 178490 4mBz acid 1.467 194047 Pyridine 1 36146 2mBzP 1.367 16881 2mBz acid 1.467 11670 Pyridine The retention times for both 2mBzP and 4mBzP were similar in both HPLC data sets. The observed similarity was expected as both 2mBzP and 4mBzP shared a highly similar molecular structure. The two differ only in the position of the methoxy group; the same can also be said for both 2mBz acid and 4mBz acid in that they also share similar retention times. The respective methoxy-benzoyl phosphates and respective methoxy-benzoic acid had differing retention times. These retention time differences suggest that the phosphate group added an overall net negative charge and an increased hydrophilic nature to the molecule. The addition of a phosphate group gave the molecule the ability to travel through the column faster by making weaker interactions with the column while molecules lacking the phosphate group form stronger interactions with the column. 46 A third peak was identified in both 4mBzP and 2mBzP synthesis products at 258 nm possessed the longest retention time on the HPLC spectra. The retention time of this peak was longer than the retention times of both the methoxy-benzoyl phosphates and the methoxybenzoic acids which indicated that this molecular species was more hydrophobic. The combination of longer retention time and detection wavelength identified this peak as pyridine, a component of the reaction system. The presence of pyridine in the synthetic reaction indicates that either pyridine was too concentrated in the reaction system, or that recrystallization did not sufficiently remove pyridine from the synthesis products. Therefore, ~50% pyridine was too high of a concentration in the reaction conditions for the synthesis of both 4mBzP and 2mBzP and the amount of pyridine should be decreased in future synthesis reactions. The micro-synthesis served as the preliminary test that illustrated that the revised synthetic method sufficiently produces both 2mBzP and 4mBzP. On the micro-scale this synthetic method produced both 2mBzP and 4mBzP, the yields of each was determined based on the mass of each product. Their respective yields were 31% and 72%; both yields were greater than the expected yield of 30% that was based on the original method (8). The respective yields suggest that the revised synthetic method on the micro- scale produced more methoxy-benzoyl phosphate product than the original synthesis method. In determining the viability of the revised synthetic method in producing both 2mBzP and 4mBzP the purity of the synthetic reaction was determined. The purity of both 2mBzP and 4mBzP was assessed by investigating the presence of two main impurities, the respective methoxy-benzoic acid and inorganic phosphate. The first was confirmed as a byproduct of the revised synthetic method by HPLC, and the latter was suspected to be an impurity due to the molar excess of phosphate that was introduced by the addition of phosphoric acid. Both the respective methoxy-benzoic acids and inorganic phosphate possess the potential to interfere with the AcPase-mediated hydrolysis of carboxy-phosphate bonds. The presence of the respective methoxy benzoic acids, detected by HPLC, confirmed them as byproducts of the revised synthesis method. The methoxy-benzoic acid byproducts were produced from a side reaction between water and the respective methoxy-benzoyl chloride. The side reaction decreases the amount of available methoxy-benzoyl chloride that can react with phosphoric acid. The amount of each respective methoxy-benzoic acid produced during the synthesis reaction of 2mBzP and 4mBzP was investigated and determined by using the 47 relationship between concentration of the methoxy benzoic acid and peak height on the HPLC 70000 60000 y = 58926x + 77.438 R² = 0.99992 50000 A 40000 30000 20000 10000 0 0 Peak height 278 nm (AU) Peak height 258 nm (AU) that are presented in figure 4.9. 250000 200000 y = 219773x - 8476.8 R² = 0.99231 B 150000 100000 0.5 1 1.5 Concentration of 2mBz acid (mM) 50000 0 0.000 0.500 1.000 1.500 Concentration of 4mBz acid (mM) Figure 4.9 The standard curves that describe he relationship between the concentration of A) 2mBz acid and B) 4mBz acid concentration and HPLC peak height at 278 nm for 2mBz acid and 258 nm for 4mBz acid. The amount of each methoxy-benzoic acid byproduct present in the synthesis product was determined by using peak heights on the HPLC (figure 4.9) The detection of 2mBz acids in the 2mBzP synthesis reaction occurred at 258 nm because 2- methoxy-benzoic acid absorbs weakly at 278 nm. Detection at 278 nm will result in smaller concentrations being hard to detect, and since this wavelength is at the lower end of the detectable range of the instrument will produce inconsistency in the concentrations obtained. This trend was also observed for 4methoxy-benzoic acid; the standard curve and amount present in the synthetic reaction was determined at 278 nm as the molecule absorbs weakly at 258 nm. The 2mBzP synthesis reaction contained 0.52% 2mBz acid while the 4mBzP synthesis reaction contained 1.99% 4mBz acid. The amounts of each respective methoxy-benzoic acids indicate that the side reaction that produces them was not a major reaction during the synthesis. Thus, major reaction of the synthesis was the production of methoxy-benzoyl phosphates. The quantification of inorganic phosphate present in each 4mBzP and 2mBzP synthesis reaction was determined through using the malachite green assay (45). The malachite green assay uses a colourimetric reaction to detects the amount of inorganic phosphate present by turning an intense green-blue from yellow as the concentration of inorganic phosphate increases. The colour change is detected at 620 nm (figure 4.5). 48 y = 0.0011x + 0.1234 R² = 0.98047 Absorbance (620 nm) 0.245 0.225 0.205 0.185 0.165 0.145 0.125 0.105 0 20 40 60 80 Concentration of Pi (uM) 100 120 Figure 4.10 The malachite green assay standard curve ranging from 0-102.2 µM inorganic phosphate for determining inorganic phosphate concentrations measured at wavelength 620 nm. Performed in triplicate, average absorbance values are plotted with the standard deviation present as error bars. The amount of inorganic phosphate present in each 2mBzP and 4mBzP synthesis product indicated that excess phosphoric acid was introduced to the synthesis reaction. Inorganic phosphate is known to inhibit AcPase activity and therefore, inorganic phosphate is unwanted in the final products of the synthesis as it will impact the kinetic characterizations of Har. marismortui AcPase. The total inorganic phosphate content of the 4mBzP synthesis reaction was 36.8%, and the inorganic content of the 2mBzP synthesis product was 27.3%. The presence of excess inorganic phosphate after the synthesis illustrates that both 2mBzP and 4mBzP must be purified further before they are used to kinetically characterize Har. marismortui AcPase. The comparison of both synthetic methods, the original method as described by Paoli et al (1995) and the revised method determined which method was optimal in producing both 2mBzP and 4mBzP (Table 4.3). First, the overall percent yield of the methoxy-benzoyl phosphate products from each synthesis reaction were compared. Between the two, the original synthesis method had the lowest percent yield of 30% (8). The yield of 2mBzP produced by the revised synthesis method was similarly low (31%). The low yield of 2mBzP could be due to the molecule being more difficult to synthesize as the yield of 4mBzP by the revised method was 72.3% which was greater than either synthetic method for producing 2mBzP. Overall the revised synthetic method had an average larger yield of the methoxy-benzoyl phosphate products indicated that the revised synthetic method was the better synthesis method. 49 Table 4.3 The comparison of the original synthesis method of producing 2mBzP and the revised synthesis method of producing both 2mBzP and 4mBzP. This comparison includes the yield, the percent of the respective methoxy benzoic acid present, the percent of inorganic phosphate present and the total percent purity of each synthesis product. Method substrate %yield %mBz acid % Pi %Purity Original (8) 2mBzP 30% unreported unreported 70% 2mBzP 31% 0.52% 27.3% 72% 4mBzP 72% 2% 36.8% 62% Revised The comparison of the two synthetic methods includes the comparison of both the impurity present and the amount of each impurity that was present. The published data for the original synthesis method did not include the percentages of each impurity present before the product was purified further, only the overall purity of the final product (8). The major impurity of the revised synthetic method was inorganic phosphate while the respective methoxy-benzoic acids were minor impurities, where each comprised of less than 5% of the final product. This showed that the approach used to minimize the amount of water present in the reaction proved to successfully decrease the production of the methoxy-benzoic acid byproducts. The major impurity of both 2mBzP and 4mBzP produced by the revised synthesis method was inorganic phosphate. Synthetic product purity before purification was not determined for the original method (8) therefore, it was unclear what the percentage of desired product was in the unpurified product. The amount of inorganic phosphate present in both 2mBzP and 4mBzP synthesis reactions indicates that the amount of phosphoric acid introduced in the synthesis needs to be decreased and the final products need to be purified further. Macro-Synthesis The initial micro-synthesis indicated that the revised synthetic method needed further refinement to become the optimal method to produce a large quantity of relatively pure methoxybenzoyl phosphate products. Both the percent yield of each synthetic product and the overall purity were increased by the following four revisions that were made for the macro-scale 50 synthesis (~40 ml final reaction volume). The reaction volume and the available methoxybenzoyl chlorides present were increased while both the amounts of pyridine present and phosphoric acid present were decreased. The micro-reaction used 50% pyridine; this was decreased to 18% for the macro-reaction. The molar excess phosphoric acid that was introduced was decreased while maintaining a molar excess to ensure that there was enough phosphoric acid present to react with the methoxy-benzoic chlorides that were present. The decrease in phosphoric acid introduced also decreased the water that was present. The results of the micro-synthesis suggested that the major impurities from the revised synthesis were pyridine and inorganic phosphate. The expected major impurity based on the original synthesis method was the methoxy-benzoic acid byproducts. Pyridine has not been reported previously as a byproduct of methoxy-benzoyl phosphate synthesis and the impact that pyridine possesses on AcPase activity is unknown. Inorganic phosphate is a known inhibitor of AcPase activity. The revisions to the revised synthesis method was expected to result in a decrease of pyridine, inorganic phosphate and methoxy-benzoic acid byproducts present in the 2mBzP and 4mBzP synthesis reactions. The amount of pyridine present in the 2mBzP and 4mBzP synthetic reactions was determined by the absorbance of each synthetic product at 195 nm, one of the three wavelengths at which pyridine has maximum absorption peaks. The three wavelengths maximums that pyridine absorbs at are: 195 nm, 251 nm, and 270 nm. Two of these wavelengths are relatively close those that were used for detection of substrate 2mBzP and 4mBzP and would be unsuitable for the detection and quantification of the pyridine present in each synthesis reaction. The absence of a pyridine peak on the HPLC spectra at 278 nm indicated pyridine absorption was minimal. Because of this, 195 nm was used to assess the amount of pyridine present in the synthetic products (figure 4.11). 51 2.0 1.8 Abs @ 195 nm 1.6 1.4 1.2 y = 0.0996x + 0.2873 R² = 0.97213 1.0 0.8 0.6 0.4 0.2 0.0 0 2 4 6 8 10 12 14 16 18 Concentration of Pyridine (ng/𝜇l) Figure 4.11 The standard curve used to determine the concentration of pyridine present in both 2mBzP and 4mbzP synthetic products at 195 nm. The assay performed in triplicate and the errors bars represent the standard deviation. Pyridine was an unexpected impurity that was found during the micro-synthesis of 2mBzP and 4mBzP. The impact of this molecule on AcPase activity is currently unknown, which required that this impurity be minimalized in the final product. Overall there was a small amount of pyridine present in the synthetic products, 0.794% of 2mBzP and 0.192% of 4mBzP. The small relative amounts of pyridine in the synthetic products suggested that 18% pyridine was enough to produce the products and the majority can be purified out of the final product. Overall there was very little pyridine present in both the 2mBzP and 4mBzP samples indicating that the smaller reaction amount of pyridine was optimal in this synthesis. The amount of inorganic phosphate present in the synthetic reaction was important as inorganic phosphate is a known inhibitor of AcPase activity. The amount of inorganic phosphate was determined using the malachite green assay, as previously described (figure 4.12). 52 Absorbance (620 nm) 1.2 1.0 0.8 y = 0.0359x + 0.1368 R² = 0.97303 0.6 0.4 0.2 0.0 0 5 10 15 20 25 30 Pi cocentration (µM) Figure 4.12 The malachite green assay ranging from 0-30 µM inorganic phosphate measured at 620 nm standard curve for the determination of inorganic phosphate in 2mBzP and 4mBzP synthetic products before and after purification. Each assay was performed in triplicate and error bars are representative of standard deviation The micro-synthesis of each methoxy-benzoyl phosphate revealed that there was a large excess of inorganic phosphate present in the final products. The excess of inorganic phosphate present in the synthetic reactions suggested that the phosphoric acid introduced during the synthesis leaves a large excess of inorganic phosphate behind. In the macro-synthesis, the introduced phosphoric acid was reduced to reduce the leftover inorganic phosphate present in the final product. Before FPLC purification there was 6.16% inorganic phosphate present in the 2mBzP product and 5.28% inorganic phosphate present in the 4mBzP product. The inorganic phosphate present in both 2mBzP and 4mBzP indicated that the amount of phosphoric acid introduced during the synthesis directly influenced the leftover inorganic phosphate present in each product. After FPLC purification of 2mBzP and 4mBzP the presence of inorganic phosphate was assessed again to determine the efficiency of the purification process. After FPLC purification there was 0.0186% inorganic phosphate present in 2mBzP and 0.0159% inorganic phosphate present in 4mBzP. Column chromatography purification effectively removed nearly all the inorganic phosphate present in the final synthesis product resulting in a small amount remaining that was expected have a minimal effect on AcPase activity. 53 The micro-synthesis reaction of both 2mBzP and 4mBzP revealed that the respective methoxy benzoic acids were small byproducts of the revised synthesis method. The carboxylic acid byproducts that were produced in the macro synthesis reaction were the result of a small amount of water introduced into the reaction. The water came from the introduction of phosphoric acid. To reduce the water that was introduced, a highly concentrated phosphoric acid solution was used to maintain the high molar excess required for synthesis. The small amounts of each respective methoxy benzoic acid produced in the macro-synthesis indicated that the production of these byproducts was a minor reaction. The micro-synthesis of 2mBzP and 4mBzP revealed that chromatography-based methods were effective in separating the methoxy-benzoyl phosphates from their respective methoxybenzoic acids and pyridine products. Each synthesis product was purified by FPLC with a 5RPColigo HPLC column at 300 nm, 278 nm and 258 nm. The FPLC chromatogram illustrated in figure 4.13 is a typical example of the purification of 2mBzP. 2mBzP was reported to absorb maximally at 300 nm making it a good indicator of where 2mBzP appears in the chromatogram (7). Of the three major impurities, both pyridine and 2mBz acid were observed on the chromatogram as they absorb at the detection wavelengths used. The FPLC chromatogram of the purification of 2mBzP shows that the synthetic product was not completely pure with minor impurities present fig. 4.13). The impurities that were present were observed in small peaks. The first peak (fraction 16) on the chromatogram, was likely a small amount of excess unbound product that eluted shortly after Buffer B was introduced to the column. The second and largest peak (fraction 40- 47) corresponds to 2mBzP as there was absorption at all three wavelengths indicating there was a molecular species present in a relatively high concentration. The third peak (fraction 56), observed at 258 nm was likely 2mBz acid, as there was no absorption at 27 8nm and 300 nm. The peak also indicated a molecular species that elutes after 2mBzP as it was less polar and formed stronger interactions with the column. The final peak (fraction 74) was a short broad peak that was absorbed primarily at 258 nm. This was identified to be pyridine due to its characteristic absorption at 258 nm and lack of absorption at the other two wavelengths. Pyridine is also less polar than both 2mBzP and 2mBz acid and formed stronger interaction with the column, eluting the column later. 54 Result file: C:\...\default\5RPC\5RPCOligoAJG004 5RPCOligoAJG004:10_UV1_278nm 5RPCOligoAJG004:10_UV3_300nm 5RPCOligoAJG004:10_Conc 5RPCOligoAJG004:10_Inject 5RPCOligoAJG004:10_UV2_258nm 5RPCOligoAJG004:10_Cond 5RPCOligoAJG004:10_Fractions 5RPCOligoAJG004:10_Logbook mAU 2500 2000 1500 1000 500 0 1 3 5 7 9 11 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56 59 62 65 68 71 74 0.0 10.0 20.0 30.0 Waste 40.0 ml Figure 4.13 The FPLC chromatogram of 2mBzP purification. Legend: the blue line indicates UV absorption at 278 nm, the pink solid line indicates UV absorption at 300 nm, the red line indicates UV absorption at 258 nm, the green line indicates %Buffer B, the brown line indicates conductance, the orange dashed lines indicate the fractions collected, and the pink dashed line indicates the injection of sample. The Y axis is measured absorption in mAU and the X axis indicates run volume in ml. Chromatogram was generated in Unicorn 5.11 55 The 4mBzP synthesis product was purified by FPLC following the same methods as the purification of 2mBzP. The same detection wavelengths were used because 4mBzP is known to absorb optimally at 258 nm, whereas 4mBz acid absorbs better at 278 nm. To assess if 4mBzP absorbs at 300 nm in a similar manner as 2mBzP does, 300 nm was included in the detection wavelengths used. The FPLC chromatogram illustrated in figure 4.14 is an example of a typical purification of 4mBzP. The chromatogram of 4mBzP purification shared some similarities with the 2mBzP purification chromatogram. The chromatogram of 4mBzP shows that the synthesis products was not completely pure with minor impurities present. The first peak appears before fraction collection was initiated (retention time before 10 min) and was not present on the 2mBzP chromatogram. The absorption of this peak at all three detection wavelengths indicated this was not purely 4mBzP.The first peak was likely a combination of 4mBzP, 4mBz acid and pyridine that did not bind to the column. The second peak (fraction 23), weakly absorbed at 258 nm, and strongly absorbed at 278 nm suggesting this peak was 4mBz acid. The third peak (fraction 3340) and largest peak, possessed the strongest absorption at 258 nm with no absorption in 300 nm and weak absorption in 278 nm. The final peak (fraction 54) was a short broad peak and absorbed at 258 nm, suggesting that this peak was pyridine. The order of the peaks demonstrates the degree that each molecular species interacted with the column. Unlike the 2mBzP purification, the 4mBz acid peak eluted off the column before the 4mBzP peak, suggesting that this species formed weaker interactions with the column than 4mBzP, causing this molecule to elute first. 56 Result file: C:\...\default\5RPC\5RPCOligoAJG003 5RPCOligoAJG003:10_UV1_278nm 5RPCOligoAJG003:10_UV3_300nm 5RPCOligoAJG003:10_Conc 5RPCOligoAJG003:10_Inject 5RPCOligoAJG003:10_UV2_258nm 5RPCOligoAJG003:10_Cond 5RPCOligoAJG003:10_Fractions 5RPCOligoAJG003:10_Logbook mAU 1400 1200 1000 800 600 400 200 0 1 3 5 7 9 11 14 17 20 23 26 29 32 35 38 41 44 47 50 53 0.0 10.0 20.0 30.0 Waste 40.0 ml Figure 4.14 The FPLC chromatogram of 4mBzP purification. Legend: the blue line indicates UV absorption at 278 nm, the pink solid line indicates UV absorption at 300 nm, the red line indicates UV absorption at 258 nm, the green line indicates %Buffer B, the brown line indicates conductance, the orange dashed lines indicate the fractions collected, and the pink dashed line indicates the injection of sample. The Y axis is measured absorption in mAU and the X axis indicates run volume in ml. Chromatogram was generated in Unicorn 5.11 57 Both substrate purification FPLC chromatograms showed that the impurities that were present were in relatively small amounts compared to the synthesis products. Both impurities, the carboxylic acid byproducts and pyridine were present in small amounts relative to the synthetic products. The individual peak sizes indicated that the FPLC purification effectively purified both 2mBzP and 4mBzP close to 100% purity. Each FPLC chromatogram had a small peak that absorbed at 258 nm, 278 nm, and 300 nm (fraction 16 and fraction 23) for 2mBzP and 4mBzP respectively, suggesting this was a combination of either unbound 2mBzP and 2mBz, or 4mBzP and 4mBz respectively. The comparison of the elution volumes for 2mBzP and 4mBzP from the FPLC purification chromatograms show that the methoxy-benzoyl phosphate products purified differently. The comparison of elution volumes of the methoxy-benzoyl phosphate products is presented in figure 4.15. The respective wavelengths used for detection were: 278 nm for 2mBzP and 258 nm for 4mBzP. Absorbance (mAU) 5000 4500 4mBzP 4000 2mBzP 3500 3000 2500 2000 1500 1000 500 0 0 10 20 30 Elution volume (ml) 40 50 Figure 4.15 The comparison of elution volumes for 2mBzP and 4mBzP during FPLC purification illustrating that the two purify differently. Detection of the methoxy-benzoyl phosphates: 278nm for 2mBzP and 258 nm for 4mBzP The comparison of the purification elution volumes showed that 2mBzP and 4mBzP interact with and elute from the column differently. The elution volume for 2mBzP was 20 ml at 65% Buffer B and the elution volume of 4mBzP was 30 ml at 80% Buffer B. The purification of 2mBzP and 4mBzP used the same buffers and followed the ten- column volume gradient ranging from 50% to 100% Buffer B. The smaller elution time indicates that 2mBzP formed weaker interactions with the column whereas the larger elution time indicates that 4mBzP formed 58 stronger interactions with the column. The differing elution volumes highlight the inherent chemical differences between 2mBzP and 4mBzP that impact purification. These inherent chemical differences arise from the location of the methoxy group and it is the comparison between these two molecules and their inherent chemical differences that give insights into the kinetic characterization of enzymes such as AcPase. To definitively determine which of the two synthetic methods was optimal in producing sufficient methoxy-benzoyl phosphate substrates for the kinetic characterization of Har. marismortui AcPase, a final comparison was required. This final comparison compiled in Table 4.4 includes the yields of each substrate and the purity before and after purification. This comparison determined that the revised synthesis method was the optimal method in that it produced more product in an overall purer product. Table 4.4 The comparison summary of the original and the revised synthesis method. This comparison includes the published values of the original method and the values obtained from the macro-synthesis of both 2mBzP and 4mBzP. Method Original (8) Molecule %yield %purity before purification %purity after purification 2mBzP 30% 70% 95% 2mBzP 47.1% 93% 99% 4mBzP 52.3% 94.5% 99% Revised The comparison of the two synthesis methods revealed that the revised method had an overall higher yield of each methoxy-benzoyl phosphate product compared to the original synthetic method which had a published yield of 30% 2mBzP, with an initial purity of 70% (8). The low yield of the original synthesis method was expected due to this being a two-step synthesis. The original synthesis first produces an intermediate before the production of the desired final product. To obtain a higher yield by this synthetic method, the first reaction (the synthesis of 2mBz anhydride) must first reach completion before proceeding on to the second step, producing 2mBzP. The revised synthesis method is a single step synthesis as it removes the first synthetic step and increases the overall yield of the reaction products. The product yields of the revised synthesis method were: 47.1% for 2mBzP and 52.3 % for 4mBzP. The overall yields 59 of 2mBzP and 4mBzP were higher than the expected yields from the micro-synthesis indicating that the micro-synthesis conditions were not ideal. With respect to the efficiency of producing 2mBzP, the revised method produced relatively more product with less starting material. The revised synthesis method overall produced products with higher purity than the products of the original synthesis method. The initial purity of both 2mBzP was 93% and 4mBzP was 94.5%, illustrating that they were relatively purer than the products of the original synthetic method. Before purification of the 2mBzP and 4mBzP synthesis products, the major impurity was inorganic phosphate. Both pyridine and the respective methoxy- benzoic acid were minor impurities of the revised synthesis method. After purification, the purity was 99% with a small amount of the inorganic phosphate remaining. The final purity of the product obtained through the original synthesis method product was 95% with a 4% impurity of inorganic phosphate and 4% of 2- methoxy-benzoic acid (8). The revised method produced methoxy- benzoyl phosphate products with both higher purity and higher relative yields indicating that this was the optimal synthetic method. BzP absorbs strongly at 283 nm while benzoic acid absorbs weakly, allowing for the hydrolysis of BzP by AcPase to be directly monitored. UV wavelength scanning of 2mBzP and 2mBz illustrated in figure 4.16 show that this trait holds true at 278 nm where 2mBzP absorbs strongly, and 2mBz absorbs weakly. The same can be said for 4mBzP, which absorbs strongly at 258 nm whereas 4mBz acid absorbs weakly. 0.9 0.9 0.8 A 0.7 0.7 0.6 0.6 0.5 0.4 2mBzP 0.3 2mBz acid Absorbance Absorbance 0.8 B 0.5 0.4 4mBzP 0.3 4mBz acid 0.2 0.2 0.1 0.1 0 0 220 270 Wavelength (nm) 320 220 240 260 280 Wavelength (nm) 300 Figure 4.16 Wave scans of A: 2mBzP and 2-methoxy-benzoic acid at 278 nm illustrating the difference in absorption B: 4mBzP and 4-methoxy-benzoic acid at 258 nm representing the difference in absorption 60 The rationale behind using substrates that contain the benzoyl groups was that the ability to directly monitor enzyme reaction progress of a kinetic reaction is was a powerful tool for studying Har. marismortui AcPase activity. Substrates containing a benzoyl group absorb in the UV spectrum, removing the necessity of finding a secondary system to the change facilitated by an enzyme. 2mBzP absorbs strongly at 278 nm while 2mBz acid absorbs weakly at this wavelength allowing the hydrolysis of 2mBzP to be directly monitored. The same pattern was observed with 4mBzP and 4mBz acid at 258 nm. This difference in absorption between respective methoxy benzoic acids and methoxy benzoyl phosphates allows reaction rates to be readily determined. Kinetic Activity The comparison between BzP, 2mBzP, and 4mBzP kinetics examined the impact of the presence and location of a methoxy group on Har. marismortui AcPase activity. The first substrate, BzP, establishes the kinetic baseline of Har. marismortui AcPase activity to which the 2mBzP and 4mBzP activity can be compared. The comparison between 2mBzP and 4mBzP allows one to investigate the impact of methoxy group placement on Har. marismortui AcPase activity. Methoxy groups are electronegative and they attract electrons towards them. When present in different locations on the benzoyl ring the methoxy group was expected to have a differing chemical impact on the carboxy-phosphate bond. The comparative kinetic study will aid in the characterization of optimal substrates for Har. marismortui AcPase. BzP Activity Assay The Har. marismortui AcPase kinetic assay with BzP established the baseline for Har. marismortui AcPase activity for a large bulky R group (the benzoyl group) present on the substrate and served as the base molecule for the comparison of the other two substrates, 2mBzP and 4mBzP. The activity of Har. marismortui AcPase with BzP produced baseline to compare impact of methoxy group position. Har. marismortui AcPase mediated hydrolysis of BzP was detected and monitored at 283 nm. The Michaelis-Menten plot constructed from a single kinetic assay investigated Har. marismortui AcPase activity with BzP is presented in figure 4.17. This representative plot was constructed using KaleidaGraph (Synergy Software) and depicts the average velocities obtained for each concentration of BzP. 61 Figure 4.17 A single assay of Har. marismortui AcPase activity with BzP, the range of 0.375- 7 mM BzP was used for the study done in triplicate. Line fit to the Michaelis- Menten equation Constructed in KaleidaGraph. Har. marismortui AcPase facilitated hydrolysis of the BzP carboxy phosphate bond followed Michaelis-Menten kinetics where there was an initial linear trend followed by a gradual plateau. Har. marismortui AcPase activity with BzP had a calculated Vmax that was 1.94x10-5 abs/sec (± 1.885x10-6), and a Km that was 1.51 mM (± 0.367). The calculated Km was within error to the expected Km value obtained from previous studies. The concentration of BzP used in this kinetic assay did not exceed 7 mM due to the limits of the spectrophotometer. Velocities obtained from assays with BzP concentrations exceeding 7 mM were inconsistent indicating that greater concentrations were outside of the linear detection range of the instrument. The calculated turnover or kcat for Har. marismortui AcPase mediated hydrolysis of BzP from the above Michaelis-Menten plot was 0.0453 s-1 (± 0.00450). From the kcat and the Km the calculated specificity constant was 0.0302 s-1mM-1 (± 0.00796). 2mBzP Activity Assay The first methoxy-benzoyl phosphate substrate used to investigate the impact of the methoxy group on Har. marismortui AcPase activity was 2mBzP. The proximity of the methoxy group to the carboxy-phosphate bond was theorized to possess the largest impact on Har. marismortui AcPase activity. Har. marismortui AcPase activity with 2mBzP was monitored at 278nm because 2mBzP absorbs strongly at this wavelength while 2mBz acid absorbs weakly 62 resulting in a negative change in absorbance. Figure 4.18 illustrates the Michaelis-Menten plot constructed from a single kinetic assay in the investigation of Har. marismortui AcPase activity with 2mBzP. This plot was constructed in KaleidaGraph (Synergy Software) and depicts the average velocities obtained for each concentration of 2mBzP. Figure 4.18 A representative kinetic assay of Har. marismortui AcPase activity with 2mBzP over the range of 0.75- 8 mM 2mBzP. Line fit to the MichaelisMenten equation in KaleidaGraph. Har. marismortui AcPase mediated hydrolysis of 2mBzP followed Michaelis-Menten kinetics as it did with BzP where there was an initial linear trend followed by a gradual plateau. Har. marismortui AcPase activity with 2mBzP had a calculated Vmax that was 1.03 x10-4 abs/sec (± 1.0876x10-5), and a Km that was 4.12 mM (± 0.847). The concentration of 2mBzP did not exceed 12 mM due to the limitations of the spectrophotometer. Velocities obtained from assays exceeding 12 mM were inconsistent indicating that the experiment was operating at the detection limits of the spectrophotometer. The calculated turnover or kcat for Har. marismortui AcPase mediated hydrolysis of 2mBzP from the above Michaelis-Menten plot was 0.1136 s-1 (± 0.0118). From kcat and Km the calculated specificity constant was 0.0277 s-1mM-1 (± 0.00636). 4mBzP Activity Assay The final methoxy-benzoyl substrate used to investigate the impact of the methoxy group on Har. marismortui AcPase activity was 4mBzP. The methoxy group is relatively further away from the carboxy group than it is with 2mBzP. The greater distance between the methoxy group and carboxy phosphate bond was theorized to have a lesser impact on Har. marismortui AcPase 63 activity. Har. marismortui AcPase activity on 4mBzP was monitored at 258 nm as 4mBzP absorbs strongly while 4mBz acid absorbs weakly resulting in a negative change in absorbance. Figure 4.19 illustrates the Michaelis-Menten plot constructed from the kinetic investigation of Har. marismortui AcPase activity with 4mBzP. This plot was constructed in KaleidaGraph (Synergy Software), plotting the average velocities obtained for each concentration of 4mBzP. Figure 4.19: A single kinetic assay of Har. marismortui AcPase activity with 4mBzP over the range of 0.33- 8 mM. Line fit to the Michaelis-Menten equation in KaleidaGraph. Har. marismortui AcPase mediated hydrolysis of 4mBzP followed Michaelis-Menten kinetics where there was an initial linear trend followed by a gradual plateau. Har. marismortui AcPase activity with 4mBzP had a calculated Vmax that was 9.51x10-5 abs/sec (± 6.18x10-6), and a Km that was 4.53mM (± 0.698). The concentration of 4mBzP did not exceed 8 mM due to limitations of the spectrophotometer. Velocities obtained from assays with concentrations greater than 8 mM were inconsistent indicating that greater concentrations were outside the limits of the machine. The calculated turnover or kcat of the hydrolysis of 4mBzP from the above MichaelisMenten plot was 0.176 s-1 (± 0.0115). From the kcat and Km, the calculated specificity constant was 0.0389 s-1mM-1 (± 0.00651). BzP, 2mBzP and 4mBzP Comparison The kinetic characterization of Har. marismortui AcPase activity was compared to determine the impact of methoxy group placement (Table 4.5). The three kinetic parameters that 64 were used in the comparison of AcPase activity with each substrate were the Km, the kcat and the specificity constant. Differences in these values gave insight into the differences in Har. marismortui AcPase activity with each given substrate as well as insight into how strong the enzyme-substrate interactions were and the efficiency of Har. marismortui AcPase. Vmax was not included in the comparison as it is relative to each reaction and is not useful in the direct comparison between each reaction unlike intrinsic values such as Km. The physical property that allows for the direct comparison between enzymatic activity with differing substrates is kcat. Table 4.5 Comparison table summarizing the Km, and kcat and specificity constant for each substrate BzP, 2mBzP, and 4mBzP where ± indicates the standard error. The comparison shows there are differences between BzP and both 2mBzP and 4mBzP in respect to Km, kcat and Specificity. Substrate Km (mM) kcat (s-1) Specificity (kcat/Km) BzP 1.5 ± 0.366 0.0453 ± 0.00450 0.0302 ± 0.00796 2mBzP 4.12 ± 0.847 0.114 ± 0.0118 0.0277 ± 0 .00638 4mBzP 4.53 ± 0.698 0.176 ± 0.0115 0.0389 ± 0.00651 The investigation into the impact of methoxy group placement required the baseline of Har. marismortui AcPase activity with BzP to be established before adding the methoxy group to the substrate. In general, the addition of the methoxy group increased the reaction rate and weakened the strength of enzyme-substrate interactions resulting in an overall more efficient reaction. Comparing 2mBzP and 4mbzP revealed that methoxy group position impacts Har. marismortui AcPase activity. Overall, Har. marismortui AcPase activity with both 2mBzP and 4mBzP resulted in a more efficient enzyme. The two methoxy- benzoyl phosphate substrates differed in the strength of the enzyme-substrate interaction. 2mBzP formed a slightly stronger interaction with Har. marismortui AcPase while in contrast 4mBzP formed a slightly weaker bond with Har. marismortui AcPase. In general, the addition of a methoxy group to the benzoyl ring increased the efficiency of Har. marismortui AcPase activity. The resulting increase in Har. marismortui AcPase efficiency arose from the electron distribution changes that were introduced from the methoxy group. The three groups of BzP are, the benzoyl group, the carboxylic acid group and the phosphate group. 65 The benzoyl group is a conjugated ring system that allows easy electron movement and the presence of other groups on the ring results in different electron distributions. The carboxylic acid group provides the substrate with an electron withdrawing group that draws in most of the electrons from around the ring and phosphate group, which increases the electron density in the relative area. Because there are groups that disrupt this electron distribution pattern, the carboxy phosphate bond is relatively strong. The placement of the methoxy group at position 2 on the benzoyl ring places the methoxy group close to the carboxy phosphate group. This position results in the electron withdrawing ‘power' doubling within the relative region of the substrate. The increase of electrons in this region decreases the electron density elsewhere. The decrease in electrons that are involved with the carboxy phosphate bond produces a chemically different carboxy bond in 2mBzP. The placement of the methoxy group at position 4 on the benzoyl ring places the methoxy group relatively far from the carboxy-phosphate bond. This position results in having two distinct electron-withdrawing centers that are relatively distant from one another that pulls the electrons in opposite directions. Position 4 is on the opposite side of the ring, and will have a lesser effect on the electron distribution of the carboxyl group with slightly fewer electrons available, but not enough to greatly impact the carboxy-phosphate bond, producing a chemically distinct carboxy-phosphate bond in 4mBzP. The strength of the carboxy-phosphate bond is reflected in the ease with which Har. marismortui AcPase hydrolyzes the bond which is described by the kcat value. Stronger carboxyphosphate bonds require AcPase to exert more energy and take more time to break, resulting in a smaller kcat. Weaker carboxy-phosphate bonds require Har. marismortui AcPase to exert less energy and take less time to break resulting in a larger kcat. Har. marismortui AcPase hydrolysis of BzP had the smallest observed kcat and the largest kcat value was associated with 4mBzP whereas the kcat value associated with 2mBzP was slightly smaller than 4mBzP. Based on the kcat values the strength of the carboxy-phosphate bond was strongest in BzP, while the carboxyphosphate bond was weakened in 2mBzP and lastly, the carboxy-phosphate bond was weakest in 4mBzP. Enzyme efficiency includes both the ease with which the enzyme acts upon a substrate and the strength of the enzyme-substrate interactions. A portion of the Har. marismortui AcPase 66 consensus sequence was speculated to interact with the substrate, orientate the substrate and bring it to the active site for hydrolysis (fig. 2.2). The residues of this loop are primarily nonpolar and organic with the ends flanked by positive residues. There is a residue in the center of the loop that varies between an aromatic residue and a positive residue. The benzoyl ring is non-polar and forms strong favourable non-polar interactions with the Har. marismortui AcPase interacting loop. Previous studies, as well as the primary kinetic assay with BzP, illustrated that the benzoyl ring is not too bulky to interact with Har. marismortui AcPase. This data supported the speculation that this single loop interacts with the substrate and pushes it towards the two catalytic residues that make up the Har. marismortui AcPase active site (fig. 2.2). The placement of the methoxy group at position 2 places a negative polar group relatively close to the carboxy phosphate bond and closer to the end of the interacting loop. This methoxy group location results in unfavourable interactions between largely non-polar residues of the interacting loop. Weak favourable interactions are capable to form between the methoxy group and a positive residue at the end of the interacting loop. The interactions between 2mBzP and interacting loop results in the optimal orientation for carboxy-phosphate bond hydrolysis and results in a rapid release of 2mBz acid. The placement of the methoxy group at position 4 places a negative polar group in the middle of the interacting Har. marismortui AcPase loop. This methoxy group location results in unfavourable interactions between non-polar residues and steric hindrance of favourable interactions with the remainder of the ring ‘sticking out.' The methoxy group prevents strong non-polar interactions from forming between the interacting loop and the substrate. The methoxy group is close enough to a positive residue in the interacting loop to form weak stabilizing interactions. The interactions between 4mBzP and the interacting loop results in the optimal orientation for carboxy-phosphate bond hydrolysis and results in a rapid release of 4mBz acid. Each of the substrates BzP, 2mBzP and 4mBzP, interact with Har. marismortui AcPase in a different manner, which resulted in a range of binding affinities to Har. marismortui AcPase. BzP lacks groups that stick out, and any additional polar groups provide the best surface for Har. marismortui AcPase to interact with. Favourable interactions between Har. marismortui AcPase and BzP are reflected in a resulting small Km value. The comparison between 2mBzP and 4mBzP 67 indicates that the methoxy group in position 2 forms slightly more favourable interactions because the 2mBzP Km value was slightly smaller than the 4mBzP Km value. The standard error of each Km value indicates that the 2mBzP Km value overlaps with the 4mBzP Km value. The overlap in standard error indicates that the 2mBzP and 4mbzP Km values were not substantially different from one another. The 2mBzP and 4mBzP Km values are 4- fold higher than the BzP Km values and the standard error of both methoxy-benzoyl phosphate products does not overlap with BzP indicates that the 2mBzP and 4mBzP Km values substantially different from that of BzP. The specificity constant is a measure that is used for the comparison of different substrates to determine which is optimal for a given enzyme. The larger the specificity constant with a given substrate, the more the enzyme prefers that substrate. Out of the three substrates tested (BzP, 2mBzP and 4mBzP), 4mBzP had the highest specificity constant indicating that it was the most optimal substrate for Har. marismortui AcPase. Therefore, position 4 on the benzoyl ring is the optimal chemical location that Har. marismortui AcPase prefers for a methoxy group. The preference of Har. marismortui AcPase methoxy group position indicates that the AcPase native substrate likely contains an electron-withdrawing group in a similar positon to interact with the AcPase interacting loop as the methoxy group of 4mBzP does. The specificity constant for BzP was the smallest of the three substrates which indicates that the native substrate is not a largely non-polar molecule as BzP was not an optimal substrate for Har. marismortui AcPase. The results of the investigation of Har. marismortui AcPase activity with BzP, 2mBzP and 4mBzP indicate the native substrate of Har. marismortui AcPase likely has a polar electronwithdrawing group on it that acts similarly as the methoxy group does on 4mBzP by preventing the formation of strong favourable non-polar interactions between Har. marismortui AcPase and substrate. The small differences in kcat values between 4mBzP and 2mBzP indicates that the location of the methoxy group on the benzoyl group has an overall limited impact on Har. marismortui AcPase efficiency. The polar group on the native substrate is likely relatively close to the carboxy-phosphate bond to increase the overall reaction rate. 68 Acetate Inhibition of AcPase The common buffer system used for Har. marismortui AcPase kinetic investigations contains acetate (1, 2, 3, 6, 7 and 8). Preliminary studies revealed that acetate had an inhibitory effect on Har. marismortui AcPase activity. The inhibitory impact of Har. marismortui AcPase was further investigated by using BzP and monitoring hydrolysis at 283 nm. A total of five concentrations of acetate (0, 50, 100, 200 and 400 mM) and six concentrations of BzP (0.275, 0.5, 0.75, 1.5, 3 and 4.6 mM) were used. The Lineweaver- Burk plot below (figure 4.20) clearly illustrates the inhibitory nature of acetate on Har. marismortui AcPase. 120000 1/V (min/DA283) 100000 80000 0 50 60000 100 40000 200 400 20000 0 0.00 0.50 1.00 1.50 1/[S] (mM-1) 2.00 2.50 3.00 Figure 4.20 The Lineweaver- Burk plot constructed to illustrate the inhibitory effect of acetate on Har. marismortui AcPase hydrolysis of BzP. The concentrations of acetate used ranged from 0- 400 mM. Each assay was completed in triplicate and the reaction monitored at 283 nm. The inhibitory impact of acetate on Har. marismortui AcPase activity was competitive. Acetate competed with the substrate (BzP) for the Har. marismortui AcPase active site, forming an enzyme-inhibitor (EI) complex. The EI complex formed a dead-end complex and did not lead to product as it prevents the binding of the BzP to Har. marismortui AcPase. This investigation showed the common use of acetate buffers in Har. marismortui AcPase kinetic assays introduced an inhibitor to the kinetic system, in this case, a competitive inhibitor. The introduction of a competitive inhibitor produced a negative impact on how long it took Har. marismortui AcPase to reach Vmax which impacted the calculated Km values, and resulted in the inaccurate 69 characterization of Har. marismortui AcPase activity. The Vmax for the acetate- inhibited reaction was calculated to be 1.43x10-4 (± 1.023x10-5) abs/s, the Km was 2.62 (± 0.388) mM and the Ki of acetate inhibition was 482.73 (± 109) mM. AcPase Reversibility Investigation The proposed mechanism presented by Stefani et al (1997) suggested that Har. marismortui AcPase facilitates the hydrolysis of carboxy-phosphate bonds in a reversible reaction (figure 2.4). In the presence of excess benzoic acid and inorganic phosphate, Har. marismortui AcPase may function in the direction of BzP formation. To investigate Har. marismortui AcPase activity in the reverse direction, the concentration of inorganic phosphate was varied ((5, 30, 50 and 100mM) while the concentration of benzoic acid (5 mM) was held constant. The reaction was monitored at 283 nm where an increase in absorption indicated BzP formation. The initial concentrations of phosphate used in the reverse kinetics assay resulted in mixing which inflated the absorption values, requiring a set of corrections to be conducted. The raw velocities and corrected values were plotted in a single plot, illustrated in figure. 4.21. Absorbance (𝝁Au)( 283nm) 350 300 250 200 150 100 50 0 -50 0 20 40 60 80 100 120 -100 -150 Phosphate [mM] Raw values Corrected values Figure 4.21 The curve assessing Har. marismortui AcPase activity in the reverse direction. Both the raw absorbance values and the corrected values are included. Varying inorganic phosphate concentrations were used with BzP was held constant. Minimal Har. marismortui AcPase activity was observed in the reverse direction in which BzP was formed from benzoic acid and inorganic phosphate. Minimal observed Har. 70 marismortui AcPase activity in the reverse direction indicates that the reverse reaction was highly unfavourable under our experimental conditions and that most of the BzP that was formed was immediately converted into benzoic acid and inorganic phosphate. The only observable trend was that as inorganic phosphate concentrations increased, the formation of BzP decreased further suggesting that inorganic phosphate acts as an inhibitor of Har. marismortui AcPase activity. The minimal observed activity combined with inorganic phosphate inhibition results in Har. marismortui AcPase not functioning in the reverse direction. Carbamoyl phosphate Activity Investigation The N-terminus of HypF facilitates the conversion of cyanide and carbon monoxide from carbamoyl phosphate (4). The N-terminus of HypF shares sequence homology with AcPase including the AcPase consensus sequence (4). Despite these similarities, AcPase has not been characterized or reported as possessing HypF-N activity. The first step in HypF-N mediated carbamoyl phosphate conversion into carbon monoxide and cyanide is likely the removal of the phosphate group. The investigation of Har. marismortui AcPase activity with carbamoyl phosphate as a substrate aimed to investigate the potential of Har. marismortui AcPase possessing HypF-N activity. This study investigated the Har. marismortui AcPase mediated hydrolysis of the phosphate group from carbamoyl phosphate. The activity of Har. marismortui AcPase was tested over a range of carbamoyl phosphate concentrations (0, 0.1, 0.75, 1.5, 3, 6 and 10 mM). Two detection assays were performed to detect the progress of the reaction: the hydroxamate assay (46) and the malachite green assay (45). The hydroxamate assay was unable to indicate presence of the carbamoyl phosphate, thus was not useful in quantifying the progress of Har. marismortui AcPase activity with carbamoyl phosphate. The malachite green assay detected the presence of free phosphate and could indicate Har. marismortui AcPase activity with carbamoyl phosphate. The results of a single kinetic assay investigating Har. marismortui AcPase activity with carbamoyl phosphate was fit to the Hill equation and is presented in figure 4.22. 71 Figure 4.22 A single kinetic assay of Har. marismortui AcPase activity with carbamoyl phosphate over a range 0- 3 mM, Activity detected by concentration of inorganic phosphate present by a malachite green assay. Data fit using the Hill equation in Kalidograph (Synergy Software). Har. marismortui AcPase did hydrolyze the phosphate from carbamoyl phosphate in the cooperative manner that is depicted in figure 4.22. Each concentration of carbamoyl phosphate required an individual negative that was run alongside the enzymatic reaction. The malachite green assay also reacted with carbamoyl phosphate producing a background reaction that needed to be accounted for. This background reaction was likely a side reaction that occurred between the reagents of the assay and carbamoyl phosphate. Har. marismortui AcPase mediated hydrolysis of carbamoyl phosphate occurs in a cooperative manner which was a characteristic that has not been observed with Har. marismortui AcPase previously. These results suggest that two HypF-N domains come together in order to facilitate carbamoyl phosphate conversion into carbon monoxide and cyanide. Interestingly, the apparent cooperative activity of Har. marismortui AcPase in vitro could be a consequence of the halophilic environment, or might suggest that AcPase also potentially functions cooperatively under physiological conditions, possibly representing an undiscovered characteristic for AcPase. 72 Chapter 5 Conclusions and Further Studies The kinetic investigation of Har. marismortui AcPase with the synthetic substrates BzP, 2mBzP and 4mBzP characterized likely properties of the native substrate for AcPase. This study investigated the impact of both the presence and location of a methoxy group on the benzoyl ring of the substrates that were used. The comparison of both the Km values and kcat values calculated for Har. marismortui AcPase activity with each substrate were used to characterize enzymesubstrate interactions. Enzyme- substrate interactions further characterized the Har. marismortui AcPase native substrate. The loop contained in the Har. marismortui AcPase consensus sequence was speculated to interact with the substrate molecule and orient the molecule so that hydrolysis of the carboxyphosphate bond can occur. The absence, presence and location of the methoxy-group resulted in these molecules interacting with the Har. marismortui AcPase interacting loop differently. The interacting loop is composed of primarily non-polar residues and forms strong favourable interactions with BzP. This results in the enzyme requiring taking more time to act upon the substrate, release the product and interact with a new substrate molecule. The introduction of the methoxy group at either position 2 or 4 interrupts the potential strong favourable interactions while providing unfavourable interactions between non-polar residues and the polar methoxy group. The addition of the methoxy group results in the enzyme requiring less work to act upon the substrate, release the substrate and interact with a new substrate molecule. These results suggest that the native substrate of Har. marismortui AcPase is not a large non-polar molecule and contains a polar group that interacts with AcPase in a similar manner of preventing the formation of strong favourable interactions. The prevention of strong favourable interactions allows the enzyme function in a more efficient manner. Further kinetic studies are required to investigate the interactions between the substrate and the Har. marismortui AcPase interacting loop. Kinetic investigations of Har. marismortui AcPase activity with 3- methoxy-benzoyl phosphate (3mBzP) will reveal the impact of the methoxy group position and if this position is more favourable than position 4. The assay with 3mBzP is expected to have a kcat and a Km value that falls between that of 2mBzP and 4mBzP due to the position of the group on the benzoyl ring. The expected results will support the findings 73 that the AcPase interacting loop does interact with the substrate and an electron withdrawing group increases the kcat value and overall efficiency of enzyme activity. Kinetic assays with a methyl group at each position around the benzoyl ring will investigate the interactions between substrate and enzyme and support the conclusions of the enzyme-substrate interactions from previous kinetic assays. The methyl group protruding out of the ring will interfere with the loop, as well as form favourable non-polar interactions with the loop. The protruding group will sterically hinder residues from forming strong favourable interacts and prevents the entire loop from interacting, while forming weaker interactions as the group is non-polar. The methyl group position on the benzoyl ring is expected to minimally impact the kcat value while the kcat value is expected to decrease closer to the carboxy-phosphate bond or at position 2 as these positions place the methyl group the furthest from the interacting loop and is expected to have minimal impact on enzyme-substrate interactions. Kinetic assays with an ammonium group at each position around the ring will investigate the impact of the group and position on enzymatic rates of Har. marismortui AcPase. The ammonium group is a stronger electron withdrawing group than the methoxy group and is expected to have a larger impact on reaction rates. This group possesses a stronger pull on electrons than the pull that methoxy groups and the carboxylic acids possess. The kcat value is anticipated to follow the same trend as it does with the methoxy group in that it is greatest at position 4 and smallest at position 2. The investigation into the inhibitory effect of acetate on Har. marismortui AcPase activity revealed that acetate acts a competitive inhibitor. Acetate competes with substrate for the Har. marismortui AcPase active site. This interaction does not lead to the production of inorganic phosphate or the carboxylic acid derivative of the substrate. Therefore, acetate is an unsuitable buffer system for studying Har. marismortui AcPase activity regardless of the substrate used. Har. marismortui AcPase is promiscuous in nature and lacks a substrate selection system. Acetate is similar enough to a natural substrate that Har. marismortui AcPase can act upon it. Har. marismortui AcPase is unable to act upon acetate binding as occupies the active site effectively inactivating the enzyme until the acetate molecule is released. Further studies on the inhibition of Har. marismortui AcPase should also investigate benzoate as a potential inhibitor as benzoate and acetate are both products of Har. marismortui AcPase mediated 74 hydrolysis. This investigation will support the results of this study and show that Har. marismortui AcPase is inhibited by the carboxylic acid derivatives of substrates that Har. marismortui AcPase acts on. The results of this study, combined with the competitive nature inorganic phosphate has on Har. marismortui AcPase shows that Har. marismortui AcPase is subject to product level inhibition that functions as a means of regulation of Har. marismortui AcPase activity. The reversibility of Har. marismortui AcPase was assessed by investigating BzP formation from benzoate and inorganic phosphate. Formation of BzP by Har. marismortui AcPase was highly unfavourable suggesting that Har. marismortui AcPase functions optimally in the direction of carboxy-phosphate bond hydrolysis. The observed trend that as phosphate concentrations increased the activity decreased supports previous studies into inorganic phosphate inhibition. Inorganic phosphate is suggested to be a competitive inhibitor in that it interacts with the active site and stalls Har. marismortui AcPase. Further studies into the reversibility of AcPase should include using smaller concentrations of both inorganic phosphate and benzoic acid to characterize the activity of Har. marismortui AcPase in the reverse direction. Characterization of the reverse reaction kcat and the Km values will provide further characterization of the Har. marismortui AcPase catalytic mechanism. The comparison between the forward and reverse reactions will support k2, the hydrolysis of the carboxy-phosphate bond, is the slowest step of the reaction. The investigation of Har, marismortui AcPase activity with carbamoyl phosphate showed that Har. marismortui AcPase catalyzes the removal of the phosphate group from carbamoyl phosphate. The activity exhibited by Har. marismortui AcPase with carbamoyl phosphate suggested that AcPase possesses HypF-N activity. The results also suggested that the removal of phosphate is a likely first step in the conversion of carbamoyl phosphate into carbon monoxide and cyanide ions by HypF-N. To further characterize Har. marismortui AcPase activity with carbamoyl phosphate a better method to detect and monitor the progress of the reaction is required. The characterization of Har. marismortui AcPase activity with carbamoyl phosphate will allow for further investigation of the cooperativity that Har. marismortui AcPase exhibits with carbamoyl phosphate. Investigations into HypF-N activity with either acetyl phosphate or BzP will show how AcPase and HypF-N compare to one another and verify that Har. marismortui AcPase exhibits HypF-N like activity. 75 Har. marismortui AcPase functions in high KCl concentrations and the kinetic characterizations may differ from the characterizations of mesophilic AcPase isoforms. The comparison between halophilic AcPase activity and mesophilic AcPase activity will reveal the impact that high salt concentrations have on this enzyme’s activity. The strong favourable interactions observed between Har. marismortui AcPase and BzP may be a consequence of the high salt conditions. Both 2mBzP and 4mBzP form more interactions with the salt environment than BzP does, and subsequently these substrates form weaker interactions with Har. marismortui AcPase. The cooperative activity exhibited by Har. marismortui AcPase with carbamoyl phosphate may also be an artifact of the high salt reaction condition. In the high salt conditions required for Har. marismortui AcPase to function the carbamoyl phosphate reaction could be unfavourable. To increase the favourability of the reaction Har. marismortui AcPase functions in a cooperative manner; a manner that has not been seen previously. The comparison of Har. marismortui AcPase activity and mesophilic AcPase isoform activity with carbamoyl phosphate will indicate if this is a novel function for all AcPase isoforms or a consequence of the halophilic environment. In summary, Har. marismortui AcPase activity with 4mBzP and 2mBzP suggests that the Har. marismortui AcPase physiological substrate contains a polar electron- withdrawing group that interacts with the enzyme specifically in a similar manner as the methoxy group does in 4mBzP. Har. marismortui AcPase is characterized as a unidirectional enzyme that is competitively inhibited by acetate suggesting that carboxylic phosphate derivatives of potential substrates inhibit Har. marismortui AcPase as a means of activity regulation. The cooperative activity exhibited by Har. marismortui AcPase with carbamoyl phosphate indicates a new functional range of Har. marismortui AcPase further characterizing Har. marismortui AcPase activity and providing insight into the natural physiological substrate. 76 References 1. Paoli, P., Fiaschi, T., Cirri, P., Camici, G., Manao, G., Cappugi, G., Raugei, G., Moneti, G., and Ramponi, G. 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Springer. 82 Appendix MAH T - - - - - MA I V - - - - - MK KWSD T E V F M SK V C - - - - M SMA EGD T L I M - - A EGN T L I M - - A EGD T L V M S T AQ - - S L K MA T - - - H N V H - - - - RAH V F V - - - - RAH L K I EM L K RM Y A R V - - - - - I I AWV S- - - - VD Y E I S- - - - VD Y E I S- - - - VD Y E I S- - - - VD Y EV S- - - - C E F EV SGR V QGV Y Y R Y GR V QGV G F R Y G L V QGV G F R Y GR V QGV G F R F GK V QGV F F R F GK V QGV F F R F GK V QGV F F R F GR V QGV C F R F GR V QGV N F R A T T R E RAQDQ WSMQ R EA R K L K F VQ I HA I R L Y T T Q Y EA K R L K Y T QA EGK K L KH T QA EGK K L K Y T QA EGK K L M Y T ED EA R K I RH A L R KA K T L GV N GWV RN L D GV N GWV RN L P G I KGY A KN L P G L T GY A KN L D G L V GWV QN T D G L V GWV QN T D G L V GWV QN T N GV V GWV KN T S G L RGWCMN S S D GR V EA V F EG D G S V EA V L EG D G S V E V V A EG D G S V E V V A CG QGT V QGQ L QG RGT V QGQ L QG RGT V QGQ L QG KGT V T GQ V QG RGT V KGY I EG PD AD V EAM V E D E E R V EA L I G Y E EA L SK L L E E EGQ V E K LMQ P A SK V RHMQ E P I SK V RHMQ E P V SK V R FMQ E P ED K V N SMK S R P A EMD VMK E F CH EG S E R - A WAHQG P P L - A R I KQG P P - A A WL K SGG P R SA WL E T KG S P K S WL E T RG S P K S WL E K RG S P K S WL SK V G S P S S WL R T T G S P L S NV T D V EV EY E R V T R V E V KWE EV EKVD Y S F S R V E R V L S E PH H I D RA S F H N E H I D KAN F N N E H I D RAN F N N E R I D R T N F SN E S I E K V E F S SQ D - - P EGV - D G Q - - P KG E - KG E - - Y KG E F ED H - - P SG E L T D KV I V K LD Y T D K V I L K L D Y SD K I I SN L D Y SD K T I SK L E Y SN R E RD R Y GY AN F EV R- - - - - - - - - - - - - - - FR I V- - - - - - - - - - - - - - - F ET Y - - - - - - - - - - - - - - - FR I R- - - - - - - - - - - - - - - FQ I V - - - - - - - - - - - - - - - FQ I V - - - - - - - - - - - - - - - FQ I I - - - - - - - - - - - - - - - F S I R- - - - - - - - - - - - - - - F H I K PD PH EN R P V H EG L G S S - - - - - -W - - - - - - G - - - - - - - - - - - - - - - - - - K - - - - - - K - - - - - - K - - - - - - Y S SH H D SN ,-./01!2"3!451!67-89!6:-;!<1=/18:1!>?2@4!A097!6!068.1!9A!2:B6<1!-<9CD71