The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre

Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a k_cat of 0.8 s^-1 and a K_m of 10 µM at pH 7.5 and 20 °C. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s^-1 and a subsequent deacylation step with a rate constant of 0.81 s^-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to K_S was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 °C, the pH-dependence of k_cat was similar to that of k_cat/K_m, indicating that formation of the acyl-enzyme did not affect the pK_a values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pK_a for k_cat, which measures the rate of deacylation, are consistent with α-amino and guanidinium groups whose pK_a values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the α-amino group of the catalytic Ser^B1 and the guanidinium group of Arg^B263 are required in neutral and protonated states respectively.