Skip to the content

Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation

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.

Full text not available from this repository. (Request a copy)

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: 20 Aug 2013 16:45
URI: http://usir.salford.ac.uk/id/eprint/186

Actions (login required)

Edit record (repository staff only)