Toggle Main Menu Toggle Search

Open Access padlockePrints

The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC11105: evidence for lowered pK(a) values of groups near the catalytic centre

Lookup NU author(s): Dr Richard Virden


Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


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, Results from stopped-how 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 degrees 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 alpha-amino and guanidinium groups whose pK(a) values are decreased in a non-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.

Publication metadata

Author(s): Morillas M, Goble ML, Virden R

Publication type: Article

Publication status: Published

Journal: Biochemical Journal

Year: 1999

Volume: 338

Issue: 1

Pages: 235-239

Print publication date: 01/02/1999

ISSN (print): 0264-6021

ISSN (electronic): 1470-8728

Publisher: Portland Press Ltd.


DOI: 10.1042/0264-6021:3380235


Altmetrics provided by Altmetric