Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes

Abstract

New reagents for the temporary blocking of active or accessible sulfhydryl groups of enzymes have been developed. These reagents, which are either alkyl alkanethiolsulfonates or alkoxycarbonylalkyl disulfides, rapidly and quantitatively place various RS- groups on the sulfhydryls to generate mixed disulfides. In all cases native enzymes can be regenerated with either dithiothreitol or beta-mercaptoethanol. In general the temporary blocking groups also afford total protection against normally inhibitory thiol blocking agents. When RS- groups were attached to rabbit muscle creatine kinase (EC 2.7.3.2), a trend toward lower residual activities with increasing bulk was observed. Treatment of the native creatine kinase with 14CH3HgC1 led to incorporation of greater than 1 equiv of CH3Hg- group per subunit. This CH3Hg- blocked enzyme was fully active, and the blocking group afforded no protection against iodoacetamide. These results suggest that CH3Hg- and the RS- groups are modifying two different sulhydryl groups on the enzyme. When papain (EC 3.4.4.10) was treated with excess methyl methanethiolsulfonate. complete and rapid inhibition was observed, and 1 equiv of CH3S- was incorporated/mol of active enzyme. Complete protection against normally inhibitory 5,5'-dithiobis(2-nitrobenzoic acid) was afforded by the temporary blocking group. When rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was titrated with methyl methanethiolsulfonate, two sulfhydryl groups per subunit were found to be modified, one much more rapidly than the other. If one extrapolates the initial slope of the titration curve, the inactivation of the enzyme would be complete after modification of a single cysteinyl residue per subunit.