Nagi M N, Prasad M R, Cook L, Cinti D L
Arch Biochem Biophys. 1983 Oct 1;226(1):50-64. doi: 10.1016/0003-9861(83)90270-9.
This study describes the biochemical properties of the rat hepatic microsomal NADPH-specific short-chain enoyl CoA reductase and NAD(P)H-dependent long-chain enoyl CoA reductase. Of the substrates tested, crotonyl CoA and trans-2-hexenoyl CoA are reduced by the short-chain reductase only in the presence of NADPH. The trans-2-octenoyl CoA and trans-2-decenoyl CoA appear to undergo reduction to octanoate and decanoate, respectively, catalyzed by both enzymes; 64% conversion of the C8:1 is catalyzed by the short-chain reductase, while 36% conversion is catalyzed by the long-chain enzyme. For the C10:1 substrate, 45% is converted by the short-chain reductase, while 55% is reduced by the long-chain reductase. trans-2-Hexadecenoyl CoA is a substrate for the long-chain enoyl CoA reductase only. Reduction of C4 and C6 enoyl CoA's was unaffected by bovine serum albumin (BSA), whereas BSA markedly stimulated the conversion of C10 and C16 enoyl CoA's to their respective saturated product. Reduction rates as a function of microsomal protein concentration, incubation time, pH, and cofactors are reported including the apparent Km and Vmax for substrates and cofactors. In general, the apparent Km's for the substrates ranged from 19 to 125 microM. The apparent Vmax for the short-chain enoyl CoA reductase was greatest with trans-2-hexenoyl CoA, having a turnover of 65 nmol/min/mg microsomal protein, while the apparent Vmax for the long-chain enzyme was greatest with trans-2-hexadecenoyl CoA, having a turnover of 55 nmol/min/mg microsomal protein. With respect to electron input, NADPH-cytochrome P-450 reductase, either alone, mixed with phospholipid, or incorporated into phospholipid vesicles, possessed no enoyl CoA reductase activity. Cytochrome c did not affect the NADPH-dependent conversion of the trans-2-enoyl CoA. In addition, anti-NADPH-cytochrome P-450 reductase IgG did not inhibit the reduction of trans-2-hexadecenoyl CoA in hepatic microsomes. Finally, the NADPH-specific short-chain and NAD(P)H-dependent long-chain enoyl CoA reductases were solubilized and completely separated from NADPH-cytochrome P-450 reductase by employing DE-52 column chromatography. These studies demonstrate the noninvolvement of NADPH-cytochrome P-450 reductase in either the short-chain (13) or long-chain enoyl CoA reductase system. Thus, the role of NADPH-cytochrome P-450 reductase in the microsomal elongation of fatty acids appears to be at the level of the first reduction step.
本研究描述了大鼠肝脏微粒体中NADPH特异性短链烯酰辅酶A还原酶和NAD(P)H依赖性长链烯酰辅酶A还原酶的生化特性。在所测试的底物中，巴豆酰辅酶A和反式-2-己烯酰辅酶A仅在NADPH存在的情况下被短链还原酶还原。反式-2-辛烯酰辅酶A和反式-2-癸烯酰辅酶A似乎分别在两种酶的催化下被还原为辛酸和癸酸；C8:1的64%转化由短链还原酶催化，而36%的转化由长链酶催化。对于C10:1底物，45%由短链还原酶转化，而55%由长链还原酶还原。反式-2-十六碳烯酰辅酶A仅是长链烯酰辅酶A还原酶的底物。C4和C6烯酰辅酶A的还原不受牛血清白蛋白（BSA）的影响，而BSA显著刺激C10和C16烯酰辅酶A向其各自饱和产物的转化。报告了还原速率作为微粒体蛋白浓度、孵育时间、pH和辅因子的函数，包括底物和辅因子的表观Km和Vmax。一般来说，底物的表观Km范围为19至125 microM。短链烯酰辅酶A还原酶的表观Vmax在反式-2-己烯酰辅酶A存在时最大，微粒体蛋白的周转率为65 nmol/min/mg，而长链酶的表观Vmax在反式-2-十六碳烯酰辅酶A存在时最大，周转率为55 nmol/min/mg微粒体蛋白。关于电子输入，NADPH-细胞色素P-450还原酶单独、与磷脂混合或掺入磷脂囊泡中均不具有烯酰辅酶A还原酶活性。细胞色素c不影响反式-2-烯酰辅酶A的NADPH依赖性转化。此外，抗NADPH-细胞色素P-450还原酶IgG不抑制肝脏微粒体中反式-2-十六碳烯酰辅酶A的还原。最后，通过DE-52柱色谱法将NADPH特异性短链和NAD(P)H依赖性长链烯酰辅酶A还原酶溶解并与NADPH-细胞色素P-450还原酶完全分离。这些研究表明NADPH-细胞色素P-450还原酶不参与短链（13）或长链烯酰辅酶A还原酶系统。因此，NADPH-细胞色素P-450还原酶在微粒体脂肪酸延长中的作用似乎处于第一个还原步骤的水平。
