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/Km, kcat and kcat/Km. 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 S3 sub-site had a small effect on Km. The binding of L-Leu to the S2 sub-site increased kcat without changing the value of Km. The analysis of the kinetic parameters revealed that, in the binding of L-Leu to the S2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil. hpbm@farmacia.ufmg.br
Henrique P B
HP
Magalhães
Arinos
A
Magalhães
Luiz
L
Juliano
David Lee
DL
Nelson
Edyr
E
Rogana
D000813:Anilides / Q000737:chemistry*; D003435:Crotalid Venoms / Q000737:chemistry* / Q000201:enzymology / Q000302:isolation & purification; D007700:Kinetics; D012044:Regression Analysis; D013379:Substrate Specificity
D000813:Anilides; D003435:Crotalid Venoms; C072644:Lachesis venom