Selective and irreversible inhibition of glutathione reductase in vitro by carbamate thioester conjugates of methyl isocyanate.

Exposure of yeast glutathione reductase (GR) in vitro to S-(N-methylcarbamoyl)glutathione (SMG) and S-(N-methylcarbamoyl)cysteine (SMC), two carbamoylating metabolites of methylisocyanate (MIC), led to a time-dependent, irreversible loss of enzyme activity (50-90%) over a period of 3 hr. The extent of inhibition was dependent upon the concentration of these carbamate thioester conjugates (0.1 to 1.0 mM) and on the presence of NADPH (100 microM). Omission of NADPH markedly attenuated the inhibitory effects of both SMG and SMC, while oxidized glutathione (GSSG), the natural substrate of the enzyme, protected against the inhibition. Parallel experiments with the antineoplastic drug N,N'-bis-(2-chloroethyl)-N-nitrosourea (BCNU), a carbamoylating agent which is known to inhibit GR selectively, gave results that were similar to those obtained with the above conjugates. When analogs of SMG and SMC labeled with 14C in the carbamoyl group were incubated with GR, radioactivity became bound covalently to the enzyme. These findings, together with the results of kinetic experiments on the release of GSH from SMG and cysteine from SMC, suggested that while both conjugates inhibit GR by carbamoylation of an active-site thiol(s), SMG exhibits a greater affinity for the active site than SMC. In contrast to the studies with GR, SMG and SMC failed to inhibit either glutathione-S-transferase (GST) or glutathione peroxidase (GPO) enzymes in vitro. It is concluded, therefore, that these conjugates most likely inhibit GR by carbamoylating free thiol groups in the active site of this enzyme, which are absent (or inaccessible) at the active-site of GST and GPO.