Abstract
The α-chymotrypsin[EC 3.4.21.1]-catalyzed hydrolysis of peptide of the type Ac-X-Glyn-NH2 (n=0, 1, 2) (X: tryptophan (Trp), 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan (Trp(NCps)), or Nin-formyltryptophan (Trp(CHO)) as the P1 residue, and glycine being appropriately replaced by alanine) was investigated to elucidate the effect on the reactivity or binding of the secondary interaction due to the C-terminal moiety of the peptide to be cleaved. Extension of the chain in the C-terminal direction by glycine was accompanied by a decrease in kcat for substrates containing Trp and Trp(NCps), in contrast with the case of extension in the opposite direction, showing that the secondary interaction due to the main chain in the C-terminal direction affects the orientation of the sensitive bond to the catalytic residues more strongly than that of the main chain in the N-terminal direction. Addition of a methyl side chain at the P2' position decreased the reactivity with peptides containing Trp or Trp(NCps) due to large increase in Km, but increased it with the peptide containing Trp(CHO) due to considerable increase in kcat. The effect of the secondary interaction is related to the nature of the P1 residue. The interaction due to a methyl side chain at the P1' position, however, favored a suitable orientation of the scissile bond to the catalytic residues. β-Alanine at the P1' position abolished reactivity but not binding, whatever the P1 residue, possibly providing evidence that the secondary interaction in the C-terminal moiety controls the rate by affecting the orientation of the sensitive bond to the functional residues of the enzyme.
