title: Mechanism of carboxypeptidase-Y-catalysed peptide semisynthesis.
abstract: The initial rate steady-state kinetics of carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions with some alpha-N-benzoyl-L-tyrosinyl compounds has been investigated using L-valinamide as the nucleophile in aminolysis. Hydrolysis of alpha-N-benzoyl-L-tyrosinyl ethyl ester, 4-nitroanilide, and -amide has been studied in the pH range 4-9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate-determining with the ester substrate, but with the 4-nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate-determining step is apparent in the range pH 4-9. For the 4-nitroanilide and the amide substrates, the kinetic parameter, Kc/Km, is influenced by an ionizing group with a pK value of 6. Probably this is the active-site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate. Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here L-valinamide was used as model nucleophile. The analysis of the observed kinetic effects of L-valinamide on the initial rate behaviour of carboxypeptidase-Y-catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl-enzyme complex forming an enzyme-aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined. The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrate. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their Kc/Km values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.
authors.affiliation: University of Copenhagen, Department of Chemistry, Denmark.
authors.forename: U
authors.initials: U
authors.lastname: Christensen
authors.forename: H B
authors.initials: HB
authors.lastname: Drøhse
authors.forename: L
authors.initials: L
authors.lastname: Mølgaard
mesh_terms: D000215:Acylation; D000814:Aniline Compounds / Q000378:metabolism; D002268:Carboxypeptidases / Q000378:metabolism*; D002384:Catalysis; D043402:Cathepsin A; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010452:Peptide Biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D029701:Saccharomyces cerevisiae Proteins; C019498:4-nitroaniline; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase; D014633:Valine