Hu X, Zhang J W, Persson A, Rydström J
Department of Biochemistry and Biophysics, Lundberg Laboratory, Chalmers University of Technology, Göteborg, Sweden.
Biochim Biophys Acta. 1995 Apr 4;1229(1):64-72. doi: 10.1016/0005-2728(94)00187-a.
(1) Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was purified in a reconstitutively active form employing affinity chromatography on immobilized palmitoyl-Coenzyme A. Reconstituted transhydrogenase showed an active proton pumping and a stimulation of the rate of reduction of 3-acetylpyridine-NAD+ by NADPH by uncouplers. Reconstitution in the absence of a thiol-reducing agent, e.g. dithiothreitol, abolished proton pumping without affecting catalytic activity, giving a decoupled transhydrogenase. (2) Co-reconstitution of transhydrogenase with bacteriorhodopsin gave vesicles which catalyzed a 5-10-fold increased rate of reduction of thio-NADP+ by NADH in the light. The Km for NADH, but not that for thio-NADP+, decreased markedly in the light, indicating an effect of the electrochemical proton potential on the affinity of the enzyme for NADH. Inhibition by substrate derivatives in the absence or presence of light supported this conclusion. Replacement of NADH with 2'-deoxy-NADH gave a strongly sigmoidal concentration dependence, indicating an allosteric change induced by binding to the NAD(H)-site. (3) Reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, previously demonstrated to be catalyzed by both reconstituted bovine transhydrogenase and detergent-dispersed E. coli transhydrogenase, occurred at a pH below 6.5. This reaction did not pump protons. Proton pumping by 3-acetylpyridine-NAD+ plus NADPH occurred at a pH above 5.5. The two reactions were thus close to mutually exclusive, with a cross point at pH 5.8. Assuming a helix bundle structure of the membrane domain of transhydrogenase, a model is proposed involving histidine 91 of the beta subunit which previously was shown to be essential by site-directed mutagenesis. According to the model the extent of protonation of this histidine determines whether proton pumping or the NADH-3-acetylpyridine-NAD+ reaction takes place.
(1) 利用固定化棕榈酰辅酶A进行亲和层析，以重组活性形式纯化了来自大肠杆菌的质子泵烟酰胺核苷酸转氢酶。重组转氢酶表现出活跃的质子泵作用，且解偶联剂能刺激NADPH对3-乙酰吡啶-NAD⁺的还原速率。在不存在硫醇还原剂（如二硫苏糖醇）的情况下进行重组，会消除质子泵作用而不影响催化活性，从而得到一种解偶联的转氢酶。(2) 转氢酶与细菌视紫红质共重组产生的囊泡，在光照下催化NADH对硫代-NADP⁺的还原速率提高了5至10倍。光照下，NADH的米氏常数显著降低，而硫代-NADP⁺的米氏常数未变，这表明电化学质子势对酶与NADH亲和力有影响。底物衍生物在有无光照时的抑制作用支持了这一结论。用2'-脱氧-NADH替代NADH产生了强烈的S形浓度依赖性，表明与NAD(H)位点结合会诱导变构变化。(3) 先前已证明重组牛转氢酶和去污剂分散的大肠杆菌转氢酶都能催化在NADPH存在下NADH对3-乙酰吡啶-NAD⁺的还原反应，该反应在pH低于6.5时发生。此反应不泵质子。3-乙酰吡啶-NAD⁺加NADPH的质子泵作用在pH高于5.5时发生。因此这两个反应几乎相互排斥，交叉点在pH 5.8。假设转氢酶膜结构域为螺旋束结构，提出了一个模型，该模型涉及β亚基的组氨酸91，先前通过定点诱变已证明其至关重要。根据该模型，该组氨酸的质子化程度决定了是发生质子泵作用还是NADH-3-乙酰吡啶-NAD⁺反应。
