SHORT-CHAIN 3-HYDROXY-2-METHYLACYL-COA DEHYDROGENASE FROM RAT-LIVER - PURIFICATION AND CHARACTERIZATION OF A NOVEL ENZYME OF ISOLEUCINE METABOLISM
SHORT-CHAIN 3-HYDROXY-2-METHYLACYL-COA DEHYDROGENASE FROM RAT-LIVER - PURIFICATION AND CHARACTERIZATION OF A NOVEL ENZYME OF ISOLEUCINE METABOLISM
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DOI：
10.1006/abbi.1995.1388
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发表时间：
1995-08-01
影响因子：
3.9
通讯作者：
SCHULZ, H
中科院分区：
文献类型：
--
作者：
LUO, MJ;MAO, LF;SCHULZ, H
短链L-3-羟基-2-甲酰基辅酶A脱氢酶（SC-HMAD）是一种可溶性线粒体酶，从大鼠肝中经6步纯化，纯化倍数为6000倍，收率为6%。在十二烷基硫酸钠存在下，通过凝胶电泳判断纯化的酶是均一的。在变性条件下，该蛋白的分子量估计为28 kDa。在非变性条件下，该酶在Sephacryl S-200上的行为类似于分子量为66 kDa的血清白蛋白。因此，SC-HMAD似乎是由两个最可能相同的28-kDa亚基组成的二聚体。用猪心L-3-羟酰基-CoA脱氢酶（HAD）（EC 1.1.1.35）的抗体进行的免疫印迹显示，SC-HMAD和HAD是免疫学上不相关的蛋白质。SC-HMAD（而非HAD）催化L-3-羟基-2-甲基丁酰-CoA（异亮氨酸的代谢物）的NAD（+）依赖性脱氢为2-甲基乙酰乙酰-CoA。具有4、5、10和16个碳原子的酰基链的3-羟基-2-甲基酰基-CoA硫酯的相对活性分别为88、100、16和0%。未支化的3-羟基酰基-CoA硫酯也是SC-HMAD的底物，尽管如L-3-羟基-2-甲基丁酰-CoA和L-3-羟基丁酰-CoA的表观K-m值分别为5和19 μ M所证明的是较差的底物。用这两种基质观察到的最大速度是相似的。它的结论是，SC-HMAD催化异亮氨酸代谢产物2-甲基丁酰辅酶A的β-氧化过程中的第二个脱氢步骤。这种酶也可能参与天然和外源性支链羧酸的β-氧化。(C)出版社：Academic Press
Short-chain L-3-hydroxy-2-methylacyl-CoA dehydrogenase (SC-HMAD), a soluble mitochondrial enzyme, was purified 6000-fold from rat Liver in 6% yield by a six-step purification procedure. The purified enzyme was homogenous as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of this protein was estimated to be 28 kDa under denaturing conditions. Under nondenaturing conditions, the enzyme behaved on Sephacryl S-200 like serum albumin with a molecular mass of 66 kDa. Thus, SC-HMAD seems to be a dimer composed of two, most likely identical 28-kDa subunits. Immunoblotting with antibodies to pig heart L-3-hydroxyacyl-CoA dehydrogenase (HAD) (EC 1.1.1.35) revealed that SC-HMAD and HAD are immunologically unrelated proteins. SC-HMAD, but not HAD, catalyzes the NAD(+)-dependent dehydrogenation of L-3-hydroxy-2-methybutyryl-CoA, a metabolite of isoleucine, to 2-methylacetoacetyl-CoA. Relative activities with 3-hydroxy-2-methylacyl-CoA thioesters having acyl chains with 4, 5, 10, and 16 carbon atoms are 88, 100, 16, and 0%, respectively. Unbranched 3-hydroxyacyl-CoA thioesters are also substrates of SC-HMAD, although poorer ones as evidenced by apparent K-m values of 5 and 19 mu M for L-3-hydroxy-2-methylbutyryl-CoA and L-3-hydroxybutyryl-CoA, respectively. Maximal velocities observed with these two substrates were similar. It is concluded that SC-HMAD catalyzes the second dehydrogenation step during the beta-oxidation of the isoleucine metabolite 2-methylbutyryl-CoA This enzyme may also be involved in the beta-oxidation of natural and xenobiotic branched chain carboxylic acids. (C) 1995 Academic Press, Inc.