On the Reactivity of the Thiol Group of Thiolsubtilisin

1 The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH‐range the –SH group of the enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in 2 H 2 O it is concluded that a mercaptide‐imidazolium ion‐pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH. 2 Iodoacetamide reacts with the –SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo‐, chloro‐, and fluoroacetamide. This might be due to the less polar environment around the –SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo‐ and chloroacetamide in the presence of dioxane. 3 By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be established above pH · 9, whereas the overall protein structure is stable up to pH · 11. D‐2‐Bromo‐ n ‐butyramide reacts with thiolsubtilisin about 60 times as fast as D‐2‐bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala‐152 and Thr‐220 of thiolsubtilisin may form van der Waals interactions with D‐2‐bromo‐ n ‐butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D‐2‐bromopropionamide and thiolsubtilisin.