Properties of Thiol-Subtilisin

The conversion of a catalytic residue of an enzyme into a functional group with similar chemical and steric properties has been investigated in order to clarify the role of the catalytic group and its tolerance to change. The active site serine residue of the proteolytic enzyme, subtilisin, was converted into a cysteine residue by a two-step chemical process with no change in other properties of the enzyme, such as sedimentation pattern, starch gel electrophoresis pattern, amino acid composition, fluorescence, ultraviolet absorption, and chemical reactivity of tryptophan residues. A small change in the optical rotatory dispersion pattern was observed. The modified enzyme, thiol-subtilisin, was found to be inactive toward normal substrates, such as other proteins and simple esters, although a slight activity toward the labile nitrophenyl esters was observed. This inactivity of thiol-subtilisin could not be attributed to the binding of substrate, which appeared to be essentially unchanged, or to the deacylation step of the over-all reaction, which was decreased up to 100-fold. Rather, it is due to the diminished rate of the acylation step, which was undetectable. This result is contrary to the rates of model reactions of thiols and alcohols as nucleophiles. It is postulated that in the active site of the enzyme there is a critical alignment of catalytic groups and substrates which is necessary for activity. The polarizability and size of the —SH group, then, may be sufficiently greater than that of the —OH group that it perturbs this critical arrangement and prevents catalytic action.