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.
