Prasad M R, Cook L, Vieth R, Cinti D L
J Biol Chem. 1984 Jun 25;259(12):7460-7.
The present study provides evidence for a new rat liver microsomal enzyme, a short chain beta-ketoacyl (acetoacetyl)-CoA reductase, which is separate from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system. This microsomal reductase converts acetoacetyl-CoA to beta-hydroxybutyryl-CoA at a rate of 70 nmol/min/mg of protein; the enzyme has a specific requirement for NADH and appears to obtain electrons directly from the reduced pyridine nucleotide without the intervention of cytochrome b5 and its flavoprotein reductase. The apparent Km of the enzyme of the acetoacetyl-CoA was 21 microM and for the cofactor, 18 microM. The pH optimum was broad, ranging from 6.5 to 8.0. The product formed is the D-isomer of beta-hydroxybutyryl-CoA. High carbohydrate fat-free diet resulted in a small but significant (35%) increase in microsomal acetoacetyl-CoA reductase activity. The cytosol also contains this enzyme activity, measuring approximately 57% of that found in the microsomes. The mitochondrial activity which is 20-25% higher than the microsomal activity appears to be due to L-beta-hydroxyacyl-CoA dehydrogenase which converts acetoacetyl-CoA to L-beta-hydroxybutyryl-CoA. The microsomal acetoacetyl-CoA reductase activity was extracted from the microsomal membrane by 0.4 M KCl, resulting in an 8- to 10-fold purification; in addition, the long chain fatty acid elongation system was unaffected by this extraction procedure. Employing beta- hydroxyhexanoyl -CoA as a substrate, evidence is also provided for a separate dehydratase which acts on short chain substrates. Lastly, the liver microsomes had no detectable acetoacetyl-CoA synthetase or acetyl-CoA acetyltransferase activities. Hence, the possible involvement of the rat hepatic microsomal short chain beta-ketoacyl-CoA reductase, short chain beta-hydroxyacyl-CoA dehydratase, and the previously reported short chain trans-2-enoyl-CoA reductase in the hepatic utilization of acetoacetyl-CoA and in the synthesis of butyryl-CoA for hepatic lipogenesis is discussed.
本研究为一种新的大鼠肝微粒体酶——短链β-酮酰基（乙酰乙酰）-CoA还原酶提供了证据，该酶与微粒体脂肪酸链延长系统的长链β-酮酰基-CoA还原酶成分不同。这种微粒体还原酶以70 nmol/分钟/毫克蛋白质的速率将乙酰乙酰-CoA转化为β-羟基丁酰-CoA；该酶对NADH有特定需求，似乎可直接从还原型吡啶核苷酸获取电子，无需细胞色素b5及其黄素蛋白还原酶的干预。该酶对乙酰乙酰-CoA的表观Km为21 μM，对辅因子为18 μM。最适pH范围较宽，为6.5至8.0。形成的产物是β-羟基丁酰-CoA的D-异构体。高碳水化合物无脂饮食导致微粒体乙酰乙酰-CoA还原酶活性小幅但显著（35%）增加。胞质溶胶中也含有这种酶活性，约为微粒体中酶活性的57%。线粒体活性比微粒体活性高20 - 25%，这似乎是由于L-β-羟酰基-CoA脱氢酶将乙酰乙酰-CoA转化为L-β-羟基丁酰-CoA所致。微粒体乙酰乙酰-CoA还原酶活性可通过0.4 M KCl从微粒体膜中提取，纯化倍数为8至10倍；此外，长链脂肪酸延长系统不受该提取过程的影响。以β-羟基己酰-CoA为底物时，还为一种作用于短链底物的独立脱水酶提供了证据。最后，肝微粒体未检测到乙酰乙酰-CoA合成酶或乙酰-CoA乙酰转移酶活性。因此，本文讨论了大鼠肝微粒体短链β-酮酰基-CoA还原酶、短链β-羟酰基-CoA脱水酶以及先前报道的短链反式-2-烯酰-CoA还原酶在肝中乙酰乙酰-CoA利用及肝脂肪生成中丁酰-CoA合成中的可能作用。
