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Lookup NU author(s): Dr Richard Virden
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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.
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.
URL: http://dx.doi.org/10.1042/0264-6021:3380235
DOI: 10.1042/0264-6021:3380235
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