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

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 subsite, the enzyme bound the transition state configuration of the substrate/product trans formation more tightly than that of the substrate. (c) 2006 Elsevier B.V. All rights reserved.Univ Fed Minas Gerais, Fac Farm, Dept Anal Clin & Toxicol, BR-31270901 Belo Horizonte, MG, BrazilFudacao Ezequiel Dias MG, Div Pesquisa & Desenvolvimento, Belo Horizonte, MG, BrazilEscola Paulista Med, Dept Biofis, BR-04023 São Paulo, BrazilUniv Fed Minas Gerais, Fac Farm, Dept Alimentos, BR-31270901 Belo Horizonte, MG, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Fisiol & Biofis, BR-31270901 Belo Horizonte, MG, BrazilEscola Paulista Med, Dept Biofis, BR-04023 São Paulo, BrazilWeb of Scienc