Inhibition of bovine kidney .alpha.-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Abstract

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney .alpha.-ketoglutarate dehydrogenase complex. Product inhibition patterns from initial velocity studies conducted at < 10-9 M or at 1.5 .times. 10-5 M Ca2+ with NAD+, CoA, or .alpha.-ketoglutarate as the variable substrate showed that the NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2- to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 .times. 10-5 M Ca2+ than for inhibition at < 10-9 M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of .alpha.-keto[1-14C]glutarate by the resolved .alpha.-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction, whereas 15 .mu.M Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the .alpha.-ketoglutarate dehydrogenase complex by binding at allosteric sites on the .alpha.-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of .alpha.-ketoglutarate was adjusted to give matching control activities at < 10-9 M Ca2+ or 1.5 .times. 10-5 M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies established that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 [substrate concentration giving half-maximal velocity] for .alpha.-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.