Permeable membrane/mass spectrometric measurement of solvent 1H/2H, 12C/13C, and 16O/18O kinetic isotope effects associated with alpha-chymotrypsin deacylation: evidence for reaction mechanism plasticity

We have measured, by permeable membrane/mass spectrometry, the 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects (kie) associated with acyl-alpha-chymotrypsin hydrolysis and transesterification. The hydrolysis of alpha-chymotrypsinyl 2-furoate has a 12C/13C kie of approximately 1.06. Transesterification of the same acyl enzyme shows 16O/18O, 12C/13C, and solvent H2O/D2O kinetic isotope effects of 1.015 (0.003), 1.01-1.02, and 2.226 (0.007), respectively. From the temperature independence of the 16O/18O transesterification kinetic isotope effect and kinetic data reported elsewhere Wang, C.-L. A., Calvo, K. C., & Klapper, M. H. (1981) Biochemistry 20, 1401-1408], we conclude that there are two active forms of acylchymotrypsin. We also propose that formation of the tetrahedral intermediate is the rate-limiting step in both hydrolysis and transesterification and that the position of the transition state in the transesterification is closer to the starting enzyme ester while that for the hydrolytic reaction is closer to the tetrahedral intermediate. These results are discussed in terms of reaction mechanism plasticity.