title: A study of the alkaline mesentericopeptidase active site by means of peptide chloromethyl ketones.
abstract: The kinetics of inactivation of alkaline mesentericopeptidase was studied using chloromethyl ketone derivatives of amino acids and peptides. It was shown that Tos-LysCH2Cl and Tos-PheCH2Cl did not influence the enzyme activity, while the inhibitory effect of Cbz-Ala-Gly-PheCH2Cl was 35 times that of Cbz-Ala-PheCH2Cl. The dependence of the pseudo-first order rate constant of the enzyme inactivation by Cbz-Ala-Gly-PheCH2Cl on pH and temperature indicated that a group with a pK of 6.59 and deltaHi of 7.7 kcal/mol was the site of the inhibitor's attack. Amino acid analysis of the modified totally inactive enzyme revealed a definite loss of histidine and after performic acid oxidation a recovery of 3-carboxymethyl histidine. The whole set of experimental data is convincing evidence on behalf of a selective alkylation of the N-3 of the active site histidine after treatment with Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl. Alkaline mesenteriocopeptidase possesses an extended active site and only a peptide chloromethyl ketone, covering a determined sequence of theenzyme molecule (S3, S2, S1, S'1, S'2, S'3 ...) is able to provide effective inhibition. The values of the inactivation constant (kinact) for Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl are close to the corresponding values reported for subtilisin Amylosacchariticus.
mesh_terms: D000590:Amino Acid Chloromethyl Ketones; D000596:Amino Acids / Q000032:analysis; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013381:Subtilisins / Q000037:antagonists & inhibitors
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000596:Amino Acids; D011480:Protease Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013381:Subtilisins; C021553:mesentericopeptidase