Kinetic study of nucleophile specificity in dipeptide synthesis catalyzed by clostridiopeptidase B.
The kinetic parameters of Clostridiopeptidase B-catalyzed aminolysis of carbobenzoxyarginyl methyl ester leading to the formation of various dipeptides are investigated. The deacylation rates of the acylenzyme were evaluated by direct product analysis using high-performance liquid chromatography on a reversed-phase column. On the basis of the partitioning ratio and the first-order and second-order rate constants for the deacylation step, large differences in the nucleophile reactivities, which appear to be related to a S'1-P'1 interaction, were observed. The order of specificity was established as Leu much greater than Ser greater than Phe greater than Val greater than Ala = Gly much greater than Pro with second-order rate constants ranging from 578,614 M-1 s-1 for leucinamide to 5132 M-1 s-1 in the case of prolinamide. All of the amino acid amides had a nucleophilic strength at least 10 times higher than that of water during the deacylation step. The data reported here represent the first experimental evidence for the existence of a S'1 site engaged in the recognition of the amino acid side chain residue for this enzyme. The recognition site showed an increase in the affinity along with an increase in the hydrophobicity of the amino acid amide side chains.
Fortier
G
G
Département de chimie, Université du Québec, Montréal, Canada.
Gagnon
J
J
D000596:Amino Acids; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004151:Dipeptides / Q000096:biosynthesis* / Q000302:isolation & purification; D004789:Enzyme Activation; D007700:Kinetics; D013379:Substrate Specificity
D000596:Amino Acids; D004151:Dipeptides; D003546:Cysteine Endopeptidases; C019913:clostripain