Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.8.1.4 (diaphorase)
 2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.
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PMID:Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi. 226 5

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.
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PMID:Active site-specific inhibition by 1,3-bis(2-chloroethyl)-1-nitrosourea of two genetically homologous flavoenzymes: glutathione reductase and lipoamide dehydrogenase. 392 Mar 38