COMMON-TYPE ACYLPHOSPHATASE - STEADY-STATE KINETICS AND LEAVING-GROUPDEPENDENCE

A number of acyl phosphates differing in the structure of the acyl moi ety (as well as in the leaving-group pK(a) of the acids produced in hy drolysis) have been synthesized. The K-m and V-max values for the bovi ne common-type acylphosphatase isoenzyme have been measured at 25 degr ees C and pH 5.3. The values of k(cat) differ widely in relation to th e different structures of the tested acyl phosphates: linear relations hips between log k(cat) and the leaving group pK(a), as well as betwee n log k(cat)/K-m and the leaving-group pK(a), were observed. On the ot her hand, the K-m values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme-substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated n ucleophilic accepters (glycerol and methanol); nor does it catalyse (H 2O)-O-18-inorganic phosphate oxygen exchange. It seems that no phospho enzyme intermediate is formed in the catalytic pathway. Furthermore, d uring the enzymic hydrolysis of benzoyl phosphate in the presence of O -18-labelled water, only inorganic phosphate(and not benzoate) incorpo rates O-18, suggesting that no acyl enzyme is formed transiently. All these findings, as well as the strong dependence of k(cat) upon the le aving group pK(a), suggest that neither a nucleophilic enzyme group no r general acid catalysis are involved in the catalytic pathway. The en zyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The acti vation energy values for the catalysed and spontaneous hydrolysis of b enzoyl phosphate have been determined.