Mechanism of action and determination of the best substrate for a thrombin-like enzyme from Lachesis muta muta venom by regression analysis of the kinetic parameters determined with peptidyl p-nitroanilide substrates

Abstract

The kinetic behavior of a thrombin-like enzyme from Lachesis muta muta venom has been studied with 13 tripeptidyl p-nitroanilide substrates. Eight substrates were unprotected at the N terminus and were used for the regression analysis of the experimentally determined kinetic parameters 1/ K m, k cat and k cat/ K m. The individual contribution of each amino acid side chain to the kinetic parameters was calculated. The amino acid sequence of the ideal substrate ( D-Pro-Leu-Arg-pNA) was determined from a regression analysis for each kinetic parameter. This result was confirmed experimentally. The structural analysis of the tripeptides showed that the binding to the S 3 sub-site had a small effect on K m. The binding of l-Leu to the S 2 sub-site increased k cat without changing the value of K m. The analysis of the kinetic parameters revealed that, in the binding of l-Leu to the S 2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.