( Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031,China; ¹Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200032, China）

Abstract

The pH-dependence in the catalytic reaction of recombinant penicillin G acylase and its mutants from B.megaterium has been studied by using kinetic methods. pK1 and pK2 of the residues of the wild type penicillin G a cylase, invo lved in the catalyzed reaction, were 5.50--5.87 and 10.73, respectively, from th e curves of logVm and log(Vm/Km) versus pH. Results showed tha t the pK1 and pK2 values of these residues of the mutants were similar to that of the wild type. pK1 of 5.64--5.86 for mutant A and 5.69--6.96 for mutant B were obtained, while pK2 was 10.61 and 10.48 for mutant A and B, respectively. At the same time, pK values at different temperatures were investigated. The ionization e nthalpies(ΔH) were 44.38--59.03 kJ/mol and 147.37 kJ/mol, respectively, from th e curve of pK versus temperature. It was presumed according to the results mentioned above that the ionizing residues, involved in the reaction, wer e histidine and lysine that are localized around the active site.