The pH-dependent Catalytic Reaction of Penicillin G Acylase and Its Mutants

( 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.