Interactions of BPN' and Carlsberg subtilisins with peptides containing aromatic amino acids at the C-terminus. Specific rate enhancement due to the secondary enzyme-substrate interaction.

The interaction between BPN' or Carlsberg subtilisins and peptides of the type Ac-Glyn-X-OMe (n = 0, 1, 2, 3), where X denotes one of five different aromatic amino acids, was investigated to elucidate the effect of the secondary interaction on catalysis in relation to the nature of the X residue. The increase in interaction upon elongation of the chain was accompanied by a large increase in kcat but with no marked change in Km in all the series of sensitive substrates. The peptides containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, acted as competitive inhibitors and exhibited an invariant dissociation constant in spite of the different chain lengths. These observations suggest that the secondary enzyme-substrate interaction induces a conformational change in the active site of the enzyme or in the substrate in such a way as to lower the activation energy and to form a stabilized transient complex. In this respect, BPN' and Carlsberg subtilisins are similar to porcine pepsin and Streptomyces griseus protease 1 rather than to alpha-chymotrypsin.