Interaction of methylchymotrypsin with Streptomyces subtilisin inhibitor.
The effect of methylation of histidine-57 of alpha-chymotrypsin with Streptomyces subtilisin inhibitor was examined. Methylchymotrypsin was isolated by affinity chromatography on inhibitor-Sepharose, and the interaction of this inactive enzyme with inhibitor was quantitatively analyzed by two different methods: the spectrophotometric titration of difference spectrum resulted in the complex formation and the application of competitive enzyme assay by using substrates of large Km values. The former method gave values of 8.6 . 10(-6) M as dissociation constant (Kd) of methylchymotrypsin . inhibitor complex and 0.91 as the number of binding sites (n) per inhibitor monomer, both of which were almost equivalent to those for native enzyme . inhibitor complex. By the latter novel method, values of 7.9 . 10(-6) M and 1.08 were obtained for Kd and n, respectively, for interaction of inhibitor with alpha-chymotrypsin, and 8 . 10(-6) M as Kd for methylchymotrypsin . inhibitor complex. These results indicate that methylation of histidine-57 of active site in alpha-chymotrypsin molecule does not affect essentially the binding ability to inhibitor and the modified enzyme binds stoichiometrically to inhibitor, as the native enzyme does, with a molar ratio of 1:1 per inhibitor monomer.
D001426:Bacterial Proteins; D002918:Chymotrypsin / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D008433:Mathematics; D008745:Methylation; D011485:Protein Binding; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D001426:Bacterial Proteins; D006639:Histidine; D013381:Subtilisins; D002918:Chymotrypsin