title: Substrate recognition mechanism of carboxypeptidase Y.
abstract: To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu)n Ala-OH (n = 1 to 6), Fmoc-(Glu)n Ala-NH2 (1 to 5), and Fmoc-Lys(Glu)3Ala-NH2 were synthesized, and kinetic parameters for these substrates were measured. Km for Fmoc-peptides significantly decreased as peptide length increased from n = 1 to n = 5 with only slight changes in kcat. Km for Fmoc-(Glu)(5,6)Ala-OH were almost the same as one for protein substrates described previously (Nakase et al., Bull. Chem. Soc. Jpn., 73, 2587-2590). These results show that the enzyme has six subsites (S1' and S1-S5). Each subsite affinity calculated from the Km revealed subsite properties, and from the differences of subsite affinity between pH 6.5 and 5.0, the residues in each subsite were predicted. For Fmoc-peptide amide substrates, the priorities of amidase and carboxamide peptidase activities were dependent on the substrate. It is likely that the interactions between side chains of peptide and subsites compensate for the lack of P1'-S1' interaction, so the amidase activity prevailed for Fmoc-(Glu)(3,5)Ala-NH2. These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nakase
authors.forename: S
authors.initials: S
authors.lastname: Murata
authors.forename: H
authors.initials: H
authors.lastname: Ueno
authors.forename: R
authors.initials: R
authors.lastname: Hayashi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D043402:Cathepsin A; D005449:Fluorenes / Q000737:chemistry; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D005449:Fluorenes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D009842:Oligopeptides; D002268:Carboxypeptidases; D043402:Cathepsin A