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; Brocklehurst, K

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.

Citation

Reid, J., Hussain, S., Bailey, T., Sonkaria, S., Sreedharan, S., Thomas, E., …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), 699-703. https://doi.org/10.1042/BJ20031318

• Journal Article Type: Article
• Publication Date: Mar 1, 2004
• Deposit Date: Aug 8, 2007
• Journal: Biochemical Journal
• Print ISSN: 0264-6021
• Publisher: Portland Press
• Peer Reviewed: Peer Reviewed
• Volume: 378
• Issue: 2
• Pages: 699-703
• DOI: https://doi.org/10.1042/BJ20031318
• Keywords: Actinidin, cysteine proteinase mechanism, free enzyme isomerization, papain, solvent dependence of rate-determining step, uncompetitive inhibition
• Publisher URL: http://dx.doi.org/10.1042/BJ20031318