The effects of increasing the content of the aprotic dipolar organic co-solvent acetonitrile on the observed first-order rate constant (kobs) 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 kobs 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 µM. The unusual negative dependence of kobs on [S]0 in the range 25–150 µM 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.
- cysteine proteinase mechanism
- free enzyme isomerization
- solvent dependence of rate-determining step
- uncompetitive inhibition
↵1 Present address: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.
↵2 Present address: Structural Biology Section, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, U.K.
↵3 Present address: Thrombosis Research Institute, Leopold Muller Laboratory, Emmanuel Kaye Building, Manresa Road, London SW3 6LR, U.K.
- The Biochemical Society, London ©2004