Importance of the carboxyl-terminal four amino acid residues in the inhibitory activity of Streptomyces subtilisin inhibitor (with a revision of its carboxyl-terminal sequence).

Abstract

The carboxyl-terminal amino acid sequence of Streptomyces subtilisin inhibitor (SSI) was reinvestigated by analysis of the amino acid sequences of the thermolysin peptides from the C-terminal decapeptide, and the sequence -Val110-Ala-Phe-Phe113, which was reported in J. Biochem. 76, 1191-1209 (1974), was revised to -Val110-Phe-Ala-Phe113. Carboxypeptidase A digestion of SSI resulted in loss of the inhibitory activity in parallel with the release of the carboxyl-terminal four amino acid residues. The resulting modified inhibitor, des(Val110-Phe113)-SSI, possessed almost full inhibitory activity against subtilisin BPN' when the inhibitor was incubated with the enzyme in amounts less than one mol of enzyme per mol of the inhibitor. However, no inhibitor activity was observed when the molar ratio of the inhibitor to the enzyme was less than one. This phenomenon suggests that the carboxyl-terminal four amino acid residues might play an important role in the maintenance of the three-dimensional structure of SSI, which resists the action of the proteinase. The addition of more than 30-fold molar excess of SSI-(104-113)-decapeptide (C-terminal decapeptide of SSI) to the modified inhibitor resulted in refolding of the polypeptide chain, rendering it immune from proteolytic digestion.