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

Abstract

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.7-10To 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 fluor...

The Nature of General Base-General Acid Catalysis in Serine Proteases

“…The electron cloud which penetrates into the space between the hydrogen donor and acceptor atoms may help the proton to proceed rapidly in a double transfer even though the bond angles are not optimal for a single proton transfer. This proposal is strongly supported by previous observations that thiol-subtilisins do not catalyze the hydrolysis of specific substrates.8' 10, 11 Figure 1 accounts for this observationi by showing that the distance between the donor and the acceptor atoms must be larger in the thiol-enzyme if the carbonyl carbon atom approaches the nucleophile from the same direction as in the serine enzyme, which would be the case with specific substrates. It is important to note that the direction of the approach to the sulfur atom by the carbonyl carbon atom of a specific substrate is not favorable if it occurs according to the proposed model.…”

Expression in Escherichia coli and Refolding of the Malonyl-/Acetyltransferase Domain of the Multifunctional Animal Fatty Acid Synthase

“…2 It is unlikely therefore that the poor catalytic activity of the Ser-581 3 Cys mutant malonyl-/acetyltransferase results from improper folding of the protein during renaturation in vitro. In this regard the malonyl-/acetyltransferase is typical of most other members of the GXSXG family of hydrolases since replacement of the active site serine residue in subtilisin (19,20), trypsin (21), and acetylcholinesterase (22) also has produced very poor enzymes with k cat values reduced by 2-6 orders of magnitude. In the case of the malonyl-/acetyltransferase it seems likely that replacement of the active site serine with the larger cysteine atom alters the distance between the nucleophile and the catalytic His-683 since the rate of acylation of the nucleophilic residue at position 581 is significantly lowered by the Ser to Cys mutation.…”

The denaturation and degradation of stable enzymes at high temperatures

“…It is implicit in the concept of induced fit [57,58] that an enzyme must flex over a time scale in keeping with its catalytic-centre activity, and that changes between conformational substates allow catalytic activity. Enzyme activity has been demonstrated at temperatures below the glass transition [59], where mobility does not occur [60][61][62][63], but catalytic-centre activities are very low, and the significance of this activity is not yet clear.…”