A range of 4-thiaacyl-CoA derivatives has been synthesized to study the bioactivation of cytotoxic fatty acids by the mitochondrial medium-chain acyl-CoA dehydrogenase and the peroxisomal acyl-CoA oxidase. Both enzymes catalyze alpha-proton abstraction from normal acyl-CoA substrates with elimination of a beta-hydride equivalent to the FAD prosthetic group. In competition with this oxidation reaction, 4-thiaacyl-CoA thioesters undergo dehydrogenase-catalyzed beta-elimination, providing that the corresponding thiolates are sufficiently good leaving groups and can be accommodated by the active site of the enzyme. Thus, the dehydrogenase catalyzes the elimination of 2-mercaptobenzothiazole and 4-nitrothiophenolate from 4-(2-benzothiazole)-4-thiabutanoyl-CoA and 4-(4-nitrophenyl)-4-thiabutanoyl-CoA, respectively. However, the 2,4-dinitrophenyl-analogue appears too bulky and the unsubstituted thiophenyl-derivative is insufficiently activated for significant elimination. Molecular modeling shows that steric interference from the flavin ring dictates a syn rather than an anti elimination. Acryloyl-CoA, the other product of 4-thiaacyl-CoA elimination reactions, is not a significant inactivator of the medium-chain dehydrogenase. In contrast, the irreversible inactivation observed during beta-elimination using 5,6-dichloro-4-thia-5-hexenoyl-CoA (DCTH-CoA), 5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA), and 6-chloro-5,5,6-trifluoro-4-thiahexanoyl-CoA (CTFTH-CoA) is caused by release of cytotoxic thiolate products within the active site of the dehydrogenase. The double bond between C5 and C6 found in the vinylic analogues DCTH- and DCTFTH-CoA is not essential for enzyme inactivation, although CTFTH-CoA is a weaker inhibitor of the dehydrogenase. Mechanism-based inactivation with CTFTH-CoA requires elimination, is unaffected by exogenous nucleophiles, and is strongly protected by octanoyl-CoA. The peroxisomal acyl-CoA oxidase efficiently oxidizes 4-thiaacyl-CoA analogues, but is only rapidly inactivated by DCTFTH-CoA. The variable ratio of elimination to oxidation observed for DCTH-, DCTFTH-, and CTFTH-CoA may influence the metabolism of the corresponding cytotoxic alkanoic acids in vivo.

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Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)