The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: Evidence for lowered pK(a) values of groups near the catalytic centre

Abstract

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.