Substrate Recognition Mechanism of Carboxypeptidase Y

Abstract

To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu)_nAla-OH (n=1 to 6), Fmoc-(Glu)_nAla-NH₂ (1 to 5), and Fmoc-Lys(Glu)₃Ala-NH₂ were synthesized, and kinetic parameters for these substrates were measured. K_m for Fmoc-peptides significantly decreased as peptide length increased from n=1 to n=5 with only slight changes in κ_cat. K_m for Fmoc-(Glu)_5,6Ala-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 K_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)_3,5Ala-NH₂. These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network.