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Updated: Jul 15, 2026

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Published on: March 18, 2010
Acyl group transfer by proteases forming an acylenzyme intermediate: kinetic model analysis (including hydrolysis of
M Y Gololobov1, I L Borisov, V K Svedas
1A.N. Belozersky Laboratory of Molecular Biology, Moscow University, U.S.S.R.
Abstract:
The quantitative analysis of peptide synthesis via transfer of the acyl moiety from the activated donor (S) to the nucleophile (N), catalysed by proteases forming an acylenzyme intermediate, has been continued. The new kinetic model takes into account the hydrolysis of an acylenzyme-nucleophile complex (EAN). The intensity of the hydrolysis is characterized by parameter gamma equal to the ratio of the rate constant of EAN hydrolysis and the rate constant of peptide formation. The ability of the EAN complex to hydrolyse leads to a decrease in the apparent nucleophile reactivity (beta) of the aminocomponent. As a result, the maximal fractional conversions of S and N to the peptide decrease, and the apparent nucleophile reactivity becomes dependent on the nucleophile concentration. The pattern of parameter gamma influence on maximal fractional conversions depends on which component is in an excess. It is with the donor excess that hydrolysis of the EAN complex affects the peptide yield dramatically. Analytical expressions for the estimation of maximal product concentration were obtained and their accuracy evaluated.
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