Substrate recognition mechanism of carboxypeptidase Y

To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu) sub(n)Ala-OH (n = 1 to 6), Fmoc-(Glu) sub(n )Ala-NH2 (1 to 5), and Fmoc-Lys(Glu) sub(3) Ala-NH2 were synthesized, and kinetic parameters for these substrates were measured. K sub(m) for Fmoc-peptides significantly decreased as peptide length increased from n = 1 to n = 5 with only slight changes in k sub(cat). K sub(m) for Fmoc-(Glu) sub(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 Si-S5). Each subsite affinity calculated from the K sub(m) 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) sub(3,5) Ala-NH2. These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network.