Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Derivatives of l-cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l-cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k_cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k₂, arising from an increase in the transition-state free energies of acylation.