Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes.

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.     

Isosteric conversion of protein carboxyl groups into carboxamide groups. II. Application to lysozyme

For isosteric conversion of carboxyl groups of proteins into amide groups, ammonolysis of protein esters under mild conditions was attempted. Ammonolysis of methyl esters of lysozyme and bovine serum albumin proved to be incomplete. Highly reactive N-ethylsalicylamide esters of guanylated lysozyme were therefore prepared by subjecting the protein to reaction with N-ethylbenzisoxazolium ion at pH 4.2, 0 degree. Per molecule, 5-7 ester groups were introduced, with concomitant decrease of activity of 80-90%. Only 0.3 tyrosine was modified. On hydrolysis at pH 9.2 the activity was completely restored. At pH 7.9 three classes of ester groups could be distinguished: one group of high rate of hydrolysis (k1 = 1.5 min-1), three groups of intermediate rate (k2 = 0.13 min-1) and two groups of low rate (k3 = 0.018 min-1). The intermediate rate approximated the rate of hydrolysis of the model compound benzoylglycine N-ethylsalicylamide ester (k = 0.15 min-1). Ammonolysis at pH 9.2 in 2.0 M ammonia/ammonium acetate provided complete conversion of the ester groups into amide groups without restoration of activity, confirming the essentiality of certain carboxyl groups. In particular, rearrangement of the ester groups into relatively stable imide groups by O-N acyl migration was found to be completely absent. When native lysozyme was esterified with N-ethylbenzisoxazolium ion the activity did not completely return on hydrolysis.