Pig Heart Lipoamide Dehydrogenase: Solvent Equilibrium and Kinetic Isotope Effects

Betty N. Leichus, John S. Blanchard

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Abstract

Lipoamide dehydrogenase is a flavoprotein which catalyzes the reversible oxidation of dihydrolipoamide, Lip(SH)2, by NAD+. The ping-pong kinetic mechanism involves stable oxidized and two-electron-reduced forms. We have investigated the rate-limiting nature of proton transfer steps in both the forward and reverse reactions catalyzed by the pig heart enzyme by using a combination of alternate substrates and solvent kinetic isotope effect studies. With NAD+ as the variable substrate, and at a fixed, saturating concentration of either Lip(SH)2 or DTT, inverse solvent kinetic isotope effects of 0.68 ± 0.05 and 0.71 ± 0.05, respectively, were observed on V/K. Solvent kinetic isotope effects on V of 0.91 ± 0.07 and 0.69 ± 0.02 were determined when Lip(SH)2 or DTT, respectively, was used as reductant. When Lip(SH)2 or DTT was used as the variable substrate, at a fixed concentration of NAD+, solvent kinetic isotope effects of 0.74 ± 0.06 and 0.51 ± 0.04, respectively, were observed on V/K for these substrates. Plots of the kinetic parameters versus mole fraction D2O (proton inventories) were linear in all cases. Solvent kinetic isotope effect measurements performed in the reverse direction using NADH as the variable substrate showed equivalent, normal solvent kinetic isotope effects on F/KNADH when oxidized lipoamide, lipoic acid, or DTT were present at fixed, saturating concentrations. Solvent kinetic isotope effects on V were equal to 1.5-2.1. When solvent kinetic isotope effect measurements were performed using the disulfide substrates lipoamide, lipoic acid, or DTT as the variable substrates, normal kinetic isotope effects on V/K of 1.3-1.7 were observed. Using DTNB as the variable substrate, an inverse kinetic isotope effect of 0.41 ± 0.02 was observed on V/K with no solvent kinetic isotope effect on V. The solvent equilibrium isotope effect was determined to be 0.438, which allows us to calculate a fractionation factor for the thiol moieties of Lip(SH)2 of 0.526. These data suggest that, during enzyme reduction by NADH, a single proton transfer between protonated His450' and Cys45 is slowed in D2O. Oxidation of the reduced enzyme by disulfide substrates is also sensitive to solvent isotopic composition, and these data suggest that a single proton transfer between protonated His450' and the thiolate anion of the mixed enzyme-substrate disulfide is rate-limiting.

Original languageEnglish (US)
Pages (from-to)3065-3072
Number of pages8
JournalBiochemistry
Volume31
Issue number12
DOIs
Publication statusPublished - Jan 1 1992

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ASJC Scopus subject areas

  • Biochemistry

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