Lawlis V B, Roche T E
Biochemistry. 1981 Apr 28;20(9):2519-24. doi: 10.1021/bi00512a024.
Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that 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-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 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 muM 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 less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 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.
微摩尔浓度的Ca2+能显著降低NADH对牛肾α-酮戊二酸脱氢酶复合体的抑制作用[劳利斯，V. B.，& 罗奇，T. E.（1980年）《分子与细胞生物化学》32卷，第147 - 152页]。在初始速度研究中，当Ca2+浓度低于10(-9) M或为1.5×10(-5) M时，以NAD+、辅酶A或α-酮戊二酸作为可变底物进行实验，产物抑制模式表明，除了在高NAD+浓度下，NADH对这些底物中的每一种都是非竞争性抑制剂，此时双倒数图呈非线性，NADH对NAD+的抑制模式从非竞争性转变为竞争性。通过斜率和截距重绘图估计，在存在1.5×10(-5) M Ca2+时，NADH对各种底物的抑制常数比在Ca2+浓度低于10(-9) M时高2倍至12倍。这些抑制模式以及Ca2+对二氢硫辛酰胺脱氢酶组分抑制作用无影响表明，Ca2+调节的NADH抑制作用发生在变构位点，高浓度的NAD+在该位点竞争性结合。在存在5.0 mM乙醛酸（作为有效受体）的情况下，研究了纯化的α-酮戊二酸脱氢酶组分对α-酮[1-14C]戊二酸的脱羧作用。NADH（0.2 mM）或1.0 mM ATP抑制该部分反应，而15 μM Ca2+、1.0 mM ADP或10 mM NAD+刺激该部分反应并降低NADH对该反应的抑制作用。因此，这些效应物通过结合在α-酮戊二酸脱氢酶组分的变构位点来改变α-酮戊二酸脱氢酶复合体的活性。在α-酮戊二酸水平经调整后，在存在或不存在1.6 mM ADP的情况下，于低于10(-9) M Ca2+或1.5×10(-5) M Ca2+条件下进行实验，以获得匹配的对照活性，在此条件下测量了在广泛的NADH/NAD+比值范围内NADH的抑制作用。这些研究表明，在补偿Ca2+和ADP对α-酮戊二酸S0.5影响的条件下，Ca2+和ADP均降低了NADH的抑制作用。ADP在降低NADH抑制作用方面特别有效；需要进一步研究以确定这是否通过NADH和ADP在相同、重叠或相互作用的位点结合而发生。
