Abstract
The inhibitory activity of API-2c was reduced by carboxymethylation of methionine residues but not by chemical modifications of lysine and arginine residues. This indicates that API-2c requires methionine residue for inhibitory activity. Dissociation of API-2c-subtilisin BPN′ complex under mild conditions (pH-gradient dialysis) resulted in the conversion of API-2c to modified form (API-2C*).The amino acid composition of API-2c did not change upon enzymatic modification. However, modified API-2c had two NH2-terminal sequences (Asp-Ser-Pro- and Ile-Tyr-Asp-) and two COOH-termini (Phe and Met), and gave two bands on SDS-polyacrylamide gel disc electrophoresis after reduction of disulfide bonds by 2-mercaptoethanol. From these results, it is concluded that the Met-Ile bond located within a disulfide bridge in API-2c molecule is the reactive site for inhibition of subtilisin BPN′.
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