Lipoamide dehydrogenase from Escherichia coli. Steady-state kinetics of the physiological reaction.
Lipoamide dehydrogenase from Escherichia coli operates qualitatively by the same mechanism as the enzyme from pig heart. It has been suggested that quantitative differences between the two, in particular the marked inhibition of the bacterial enzyme by its product NADH, are related to the fact that the E. coli enzyme lacks the phosphorylation/dephosphorylation control present in the mammalian enzyme (Wilkinson, K. D., and Williams, C. H., Jr. (1981) J. Biol. Chem. 256, 2307-2314). Because of the inhibition by NADH, the kinetics of the E. coli enzyme have not been studied previously in the physiological direction with the natural substrate, dihydrolipoamide. We have now measured the steady-state kinetics of the oxidation of dihydrolipoamide by NAD+ using the stopped-flow technique to follow only the early time course. The pH dependence of kcat revealed an apparent pKa value of 6.7, reflecting ionization(s) of the enzyme-substrate complex. The pH dependence of kcat/Km gave an apparent pKa of 7.4 reflecting ionization(s) of the free 2-electron-reduced enzyme. The inhibition pattern for NADH was mixed, consistent with the fact that NADH is both a product inhibitor and inhibits by reducing a fraction of the enzyme to the catalytically inactive 4-electron-reduced state. There is a modest pH-dependent positive cooperativity in the saturation curve for NAD+ decreasing with increasing pH. Spectral changes in the 530 and 446 nm bands of the 2-electron-reduced enzyme, associated with the titration of the nascent thiols and the base, showed tentative pKa values of 6.4 and 7.1, respectively, in a pH jump experiment. The properties of the wild type E. coli enzyme can now be compared with those of several site-directed mutants.
Sahlman
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Veterans Administration Medical Center, Ann Arbor, Michigan 48105.
Williams
C H
CH
D000818:Animals; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation; D013053:Spectrophotometry; D013552:Swine; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D009243:NAD; C007409:dihydrolipoamide; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase