2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties.
2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties.
复制标题
来自蛔虫肌肉的 2-甲基乙酰乙酰辅酶 A 还原酶：纯化和特性。
DOI：
10.1016/0003-9861(83)90273-4
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发表时间：
1983
影响因子：
3.9
通讯作者：
Saz,HJ
中科院分区：
文献类型：
--
作者：
SuarezdeMata,Z;Zarranz,ME;Lizardo,R;Saz,HJ
2-甲基乙酰辅酶a和3-酮-2-甲基戊烷酰辅酶a被认为是蛔虫类肌肉合成2-甲基丁酸酯和2-甲基戊酸酯的中间体。这些挥发性酸是蛔虫代谢的主要发酵产物。2-甲基乙酰乙酰辅酶a还原酶从carismuscle中纯化了532倍，得到了一种均匀的制备物，在十二烷基硫酸钠存在下，在不连续聚丙烯酰胺凝胶电泳上观察到，该制备物含有单一蛋白质物种。纯化过程采用亚细胞分离、NAD+琼脂糖亲和层析和deae -纤维素离子交换层析。在纯化过程中观察到2-甲基乙酰乙酸乙酯和乙酰乙酰辅酶A的活性比恒定，表明同一酶催化了这两个反应。此外，纯化的蛋白质催化nadh依赖的3-酮-2-甲基戊酸乙酯的还原速率与2-甲基乙酰乙酸乙酯的还原速率基本相同。纯化后的酶为碱性蛋白，在4℃时等电点为8.45。天然蛋白的分子量（排斥色谱Mr= 64,000）和亚基的大小（十二烷基硫酸盐-聚丙烯酰胺电泳Mr= 30,000）表明该酶是由两个相同分子量的亚基组成的。对纯化蛋白进行的底物特异性研究表明，乙酯可以替代辅酶A衍生物，但这种替代只有在存在支化的2-甲基时才会产生活性底物。直链乙酯是无活性的。测定了底物和核苷酸的动力学常数。辅酶a酯作为蛔虫酶的生理底物的作用已得到证实。当以2-甲基乙酰乙酸乙酯为底物进行还原方向测定时，纯化酶的活性不仅被先前报道的辅酶A抑制，而且被乙酰辅酶A抑制。讨论了这些抑制的生理意义。
2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, byAscaris lumbricoidesmuscle. These volatile acids are major fermentation products ofAscarismetabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold fromAscarismuscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 °C. The molecular weight of the native protein (Mr= 64,000 by exclusion chromatography) and the size of the subunit (Mr= 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for theAscarisenzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.