Pig heart lipoamide dehydrogenase: solvent equilibrium and kinetic isotope effects.
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 V/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.(ABSTRACT TRUNCATED AT 250 WORDS)
Leichus
B N
BN
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Blanchard
J S
JS
D000818:Animals; D002384:Catalysis; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology*; D009243:NAD / Q000737:chemistry; D012997:Solvents; D013379:Substrate Specificity / Q000187:drug effects; D013552:Swine; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000737:chemistry
D004220:Disulfides; D012997:Solvents; D009243:NAD; C007409:dihydrolipoamide; D008063:Thioctic Acid; D008058:Dihydrolipoamide Dehydrogenase; D004229:Dithiothreitol