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Since the disruption of the three-dimensional structure of a protein can cause loss of enzyme activity, the interpretation of modification studies at the active sites of enzymes becomes particularly difficult. It is necessary to modify catalytic residues to establish their essentiality to the catalytic process, but any modification leading to loss in activity might also be explained as an effect on three-dimensional structure. Moreover, it is not clear how much change in the spatial position of functional groups can be tolerated by the enzyme. Although some change is apparently allowed in residues involved in binding or tertiary structure,'-3 it would be expected that enzyme activity should be even more sensitive to changes in amino acids directly involved in catalysis. Therefore, it would be desirable to modify the catalytic residues in as minor a way as possible ill order to understand the function of these residues. It has been possible to transform the serine residue at the active site of chymotrypsin to a dehydroalanine residue, a change which converted the serine to a smaller residue and thus removed any steric access ambiguities in the interpretation of the role of the serine.4 Such a modification, however, involved a considerable change in the chemical properties of the affected moiety, and it was desirable to attempt a conversion to a residue more nearly similar to that of the original serine. Such a residue is cysteine since the SH group of cysteine possesses steric and chemical properties similar to those of the OH group of serine. The van der Waals radii of oxygen and sulfur are 1.40 and 1.85 A, respectively, and the covalent radii are 0.66 and 1.04 A, respectively.6 Since the two groups react similarly in hydrolyses, such a chemical transformation would be minimal, both chemically and sterically. The SH group is, in general, somewhat more reactive than the OH group either as an attacking nucleophile or as a leaving group.7-10 To effect such a change in chymotrypsin is difficult because the chemical manipulations require the presence of active sulfide compounds which tend to break the disulfide bridges in the molecule. Therefore, subtilisin, a proteolytic enzyme with properties like those of chymotrypsin but containing no disulfide bridges, was chosen for the desired conversion. The serine at the active site of subtilisin undergoes inactivation with diisopropylfluorophosphate,'1 and the specificity of the enzyme is similar in many ways to that of chymotrypsin and other serine proteases.12 Chymotrypsin shows unusual reactivity with sulfonyl fluorides11 and this reactivity was also found to extend to subtilisin. With the use of C14-labeled phenylmethanesulfonyl fluoride, a rapid and stoichiometric inhibition of the enzyme was obtained yielding 0.95 4 0.05 mole of phenylmethanesulfonyl group per mole of subtilisin. Apparently the phenylmethanesulfonyl group reacted by a prior binding to the active site, just as in the case of chymotrypsin, since the reactivity of the similar reagent, toluene sulfonyl fluoride, was much lower with both enzymes. It seemed probable that this phenylmethainesulfonyl group in subtilisin could be

The conversion of serine at the active site of subtilisin to cysteine: a "chemical mutation".