Active site-specific inhibition by 1,3-bis(2-chloroethyl)-1-nitrosourea of two genetically homologous flavoenzymes: glutathione reductase and lipoamide dehydrogenase.
We extended our previous studies of the selectivity and mechanism of action as an enzyme inhibitor of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an antitumor drug now widely used to inactivate glutathione reductase (GSSG-R) experimentally. In contrast to other enzymes examined so far, lipoamide dehydrogenase (LSSLNH2-D) was, like its genetic relative GSSG-R, also strongly inhibited by BCNU. The drug concentration needed to inactivate GSSG-R and LSSLNH2-D was much smaller than that affecting the least resistant of five other flavoenzymes tested. When oxidized, both GSSG-R and LSSLNH2-D were resistant to BCNU, and to be effective, the drug had to interact directly with enzyme protein reduced by its specific pyridine nucleotide. In intact human erythrocytes, GSSG-R was mostly reduced and LSSLNH2-D activity undetectable. The partial genetic homology of GSSG-R and LSSLNH2-D and their special sensitivity to BCNU provided a unique opportunity to define more exactly the site of drug-enzyme interaction through comparative coenzyme studies combined with direct and reciprocal substrate competition experiments. The results, together with earlier data on the prevention of BCNU inhibition by cysteine, indicate that the nitrosourea achieves its relative selectivity against the two related flavoenzymes by interacting with at least one of the two reduced cysteinyls located within their oxidoredox active site. For GSSG-R, the attacked cysteinyl is most probably Cys-58.
D002330:Carmustine / Q000494:pharmacology*; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors*; D004347:Drug Interactions; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors*; D006801:Humans; D066298:In Vitro Techniques; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000494:pharmacology
D009249:NADP; D008063:Thioctic Acid; C013091:lipoamide; D010088:Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase; D005980:Glutathione Reductase; D002330:Carmustine