The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC11105: evidence for lowered pK(a) values of groups near thecatalytic 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 mu M at pH 7.5 and 20 degrees C, Res ults from stopped-how experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate const ant 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, m easured at pH 7.5, may be partly explained by a conformational transition a ffecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to K-s was consistent with energy-com pensation between the binding of the substrate and catalysis of the formati on of the transition state. At 20 degrees C, the pH-dependence of k(cat) wa s similar to that of k(cat)/K-m, indicating that formation of the acyl-enzy me did not affect the pK(a) values (6.5 and 9.0) of an acidic and basic gro up 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 alpha-amino and guanidinium groups whose pK(a) values are decreased in a no n-polar environment. It is proposed that, for catalytic activity, the alpha -amino group of the catalytic Ser(B1) and the guanidinium group of Arg(B263 ) are required in neutral and protonated states respectively.