Reid, JD, Hussain, S, Bailey, TSF, Sonkaria, S, Sreedharan, SK, Thomas, EW, Resmini, M and Brocklehurst, K 2004, 'Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation' , Biochemical Journal, 378 (2) , pp. 699-703.

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Abstract

The effects of increasing the content of the aprotic dipolar organic co-solvent acetonitrile on the observed first-order rate constant (k(obs)) of the pre-steady state acylation phases of the hydrolysis of N-acetyl-Phe-Gly methyl thionester catalysed by the cysteine proteinase variants actinidin and papain in sodium acetate buffer, pH 5.3, were investigated by stopped-flow spectral analysis. With low acetonitrile content, plots of k(obs) against [S]0 for the actinidin reaction are linear with an ordinate intercept of magnitude consistent with a five-step mechanism involving a post-acylation conformational change. Increasing the acetonitrile content results in marked deviations of the plots from linearity with a rate minimum around [S]0=150 microM. The unusual negative dependence of k(obs) on [S]0 in the range 25-150 microM is characteristic of a rate-determining isomerization of the free enzyme before substrate binding, additional to the five-step mechanism. There was no evidence for this phenomenon nor for the post-acylation conformational change in the analogous reaction with papain. For this enzyme, however, acetonitrile acts as an inhibitor with approximately uncompetitive characteristics. Possible mechanistic consequences of the differential solvent-perturbed kinetics are indicated. The free enzyme isomerization of actinidin may provide an explanation for the marked difference in sensitivity between this enzyme and papain of binding site-catalytic site signalling in reactions of substrate-derived 2-pyridyl disulphide reactivity probes.

Item Type:	Article
Uncontrolled Keywords:	Actinidin, cysteine proteinase mechanism, free enzyme isomerization, papain, solvent dependence of rate-determining step, uncompetitive inhibition
Themes:	Subjects / Themes > Q Science > Q Science (General) Subjects outside of the University Themes
Schools:	Colleges and Schools > College of Science & Technology Colleges and Schools > College of Science & Technology > School of Environment and Life Sciences
Journal or Publication Title:	Biochemical Journal
Publisher:	Portland Press
Refereed:	Yes
ISSN:	02646021
Depositing User:	H Kenna
Date Deposited:	08 Aug 2007 13:56
Last Modified:	27 Sep 2011 12:26
URI:	http://usir.salford.ac.uk/id/eprint/186

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