Battaile K P, McBurney M, Van Veldhoven P P, Vockley J
Mayo Clinic and Mayo Foundation, Department of Medical Genetics and Biochemistry/Molecular Biology, 200 First St, SW, Rochester, MN 55905, USA.
Biochim Biophys Acta. 1998 Feb 23;1390(3):333-8. doi: 10.1016/s0005-2760(97)00185-9.
The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes involved in fatty acid and branched chain amino-acid metabolism. Long chain acyl-CoA dehydrogenase (LCAD) and short/branched chain acyl-CoA dehydrogenase (SBCAD) have been shown to have activity towards 2-methyl branched chain acyl-CoA substrates of varying chain lengths. In humans, long chain 2-branched chain fatty acids such as pristanic acid are largely thought to be metabolized in peroxisomes through desaturation of their CoA esters by branched chain acyl-CoA oxidase, but LCAD is also capable of utilizing 2-methyldecanoyl- and 2-methylpalmitoyl-CoA as substrate [1]. Since the acyl-CoA oxidase reaction is specific for the S-enantiomer of the branched chain substrates, we investigated the stereo specificity of mitochondrial LCAD. Purified LCAD had a specific activity of 390 and 340 mU/mg of purified LCAD protein using palmitoyl-CoA and S-2-methylpentadecanoyl-CoA, respectively, as substrate. No activity was measurable with R-2-methylpentadecanoyl-CoA. Purified medium chain acyl-CoA dehydrogenase (MCAD) could also utilize S-2-methylpentadecanoyl-CoA as a substrate, but not R-2-methylpentadecanoyl-CoA. These results indicate that LCAD and MCAD are specific for the S-enantiomers of methylbranched chain substrates. Crude mitochondrial extracts showed no activity when dehydrogenating activity was measured with R/S-2-methylpalmitoyl-CoA or S-2-methylpentadecanoyl-CoA after inactivation of the extract with antibodies to very long chain acyl-CoA dehydrogenase and MCAD, suggesting that this substrate is not useful in identifyig clinical deficiencies of LCAD.
酰基辅酶A脱氢酶是一族参与脂肪酸和支链氨基酸代谢的线粒体黄素酶。长链酰基辅酶A脱氢酶（LCAD）和短链/支链酰基辅酶A脱氢酶（SBCAD）已被证明对不同链长的2-甲基支链酰基辅酶A底物具有活性。在人类中，长链2-支链脂肪酸如降植烷酸，人们普遍认为其主要在过氧化物酶体中通过支链酰基辅酶A氧化酶使其辅酶A酯去饱和来进行代谢，但LCAD也能够利用2-甲基癸酰辅酶A和2-甲基棕榈酰辅酶A作为底物[1]。由于酰基辅酶A氧化酶反应对支链底物的S-对映体具有特异性，我们研究了线粒体LCAD的立体特异性。使用棕榈酰辅酶A和S-2-甲基十五烷酰辅酶A分别作为底物时，纯化的LCAD的比活性分别为390和340 mU/mg纯化的LCAD蛋白。用R-2-甲基十五烷酰辅酶A未检测到活性。纯化的中链酰基辅酶A脱氢酶（MCAD）也可以利用S-2-甲基十五烷酰辅酶A作为底物，但不能利用R-2-甲基十五烷酰辅酶A。这些结果表明，LCAD和MCAD对甲基支链底物的S-对映体具有特异性。在用抗超长链酰基辅酶A脱氢酶和MCAD的抗体使粗线粒体提取物失活后，用R/S-2-甲基棕榈酰辅酶A或S-2-甲基十五烷酰辅酶A测量脱氢活性时，粗线粒体提取物无活性，这表明该底物对于鉴定LCAD的临床缺陷并无帮助。
