Yamaguchi M, Hatefi Y
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1993 Aug 25;268(24):17871-7.
The mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase is a homodimer of monomer M(r) = 109,065. It is composed of a 430-residue-long N-terminal hydrophilic domain, a 400-residue-long central hydrophobic domain, and a 200-residue-long C-terminal hydrophilic domain. The N- and the C-terminal hydrophilic domains extend into the mitochondrial matrix, and the central hydrophobic domain intercalates into the inner membrane. Equilibrium binding of [14C]NADH and [14C]NADPH was carried out to the purified transhydrogenase, transhydrogenase modified with N,N'-dicyclohexylcarbodiimide (DCCD) at the NADH-protectable Glu257, and to proteolytic fragments corresponding to dimers of the N-terminal and the C-terminal hydrophilic domains. The intact enzyme bound 1 mol of [14C]NADH or [14C]NADPH per dimer with Kd values of 9.5 and 5.7 microM, respectively. The 90% DCCD-inhibited enzyme bound [14C]NADPH with undiminished capacity and [14C]NADH with 10% capacity as compared with unmodified enzyme. The Kd values were unaltered. The N-terminal fragment bound [14C]NADH at 0.76 mol/dimer with unaltered Kd but did not bind [14C]NADPH. The C-terminal fragment bound 0.32 mol of [14C]NADPH/dimer with unaltered Kd but did not bind [14C]NADH. These results are consistent with our previous assignment of the NAD(H) and the NADP(H) binding sites to the N- and the C-terminal hydrophilic domains, respectively. The binding stoichiometries of 1 mol each of [14C]NADH and [14C]NADPH/dimeric transhydrogenase suggest half-of-the-sites reactivity, which is consistent with our previous findings that 100% inhibition by either p-[3H]fluorosulfonylbenzoyl-5'-adenosine or [14C]DCCD involved the incorporation of 1 mol of inhibitor/dimeric enzyme. We had shown earlier that N-(ethoxycarbonyl)2-ethoxy-1,2-dihydroquinoline (EEDQ) inhibits the transhydrogenase, apparently at the NAD binding site, but differently from DCCD. The enzyme was protected by 5'-AMP strongly against DCCD and weakly against EEDQ, whereas it was protected by NMNH strongly against EEDQ and not at all against DCCD. We have now determined the EEDQ binding sites to be Glu232 and Glu880. The different mononucleotide protections against DCCD and EEDQ suggest that the AMP moiety of NAD binds near Glu257 and its NMN portion near Glu232. They also suggest that the NMN moiety of NADP may bind near Glu880.
线粒体能量转换烟酰胺核苷酸转氢酶是一种同型二聚体，单体分子量（Mr）为109,065。它由一个430个残基长的N端亲水区、一个400个残基长的中央疏水区和一个200个残基长的C端亲水区组成。N端和C端亲水区延伸到线粒体基质中，中央疏水区插入内膜。对纯化的转氢酶、在可被NADH保护的Glu257处用N,N'-二环己基碳二亚胺（DCCD）修饰的转氢酶以及与N端和C端亲水区二聚体相对应的蛋白水解片段进行了[14C]NADH和[14C]NADPH的平衡结合实验。完整的酶每二聚体结合1摩尔[14C]NADH或[14C]NADPH，其解离常数（Kd）值分别为9.5和5.7微摩尔。与未修饰的酶相比，90%被DCCD抑制的酶结合[14C]NADPH的能力未降低，结合[14C]NADH的能力为未修饰酶的10%。Kd值未改变。N端片段每二聚体以0.76摩尔结合[14C]NADH，Kd不变，但不结合[14C]NADPH。C端片段每二聚体结合0.32摩尔[14C]NADPH，Kd不变，但不结合[14C]NADH。这些结果与我们之前分别将NAD(H)和NADP(H)结合位点定位到N端和C端亲水区的结果一致。每二聚体转氢酶结合1摩尔[14C]NADH和1摩尔[14C]NADPH的结合化学计量表明存在半位点反应性，这与我们之前的发现一致，即对-对-[3H]氟磺酰苯甲酰-5'-腺苷或[14C]DCCD的100%抑制涉及每二聚体酶掺入1摩尔抑制剂。我们之前已经表明，N-(乙氧羰基)2-乙氧基-1,2-二氢喹啉（EEDQ）抑制转氢酶，显然是在NAD结合位点，但与DCCD不同。该酶受到5'-AMP的强烈保护而免受DCCD抑制，受到较弱的保护而免受EEDQ抑制，而它受到NMNH的强烈保护而免受EEDQ抑制，完全不受DCCD抑制。我们现在已经确定EEDQ的结合位点为Glu232和Glu880。对DCCD和EEDQ的不同单核苷酸保护表明，NAD的AMP部分在Glu257附近结合，其NMN部分在Glu232附近结合。它们还表明，NADP的NMN部分可能在Glu880附近结合。
