Lee F J, Lin L W, Smith J A
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Eur J Biochem. 1989 Sep 1;184(1):21-8. doi: 10.1111/j.1432-1033.1989.tb14985.x.
Acetyl-CoA hydrolase, which hydrolyzes acetyl-CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 1080-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl-CoA hydrolase was estimated to be 64 +/- 5 kDa by gel-filtration chromatography. SDS/PAGE analysis revealed that the denatured molecular mass was 65 +/- 2 kDa and together with that for the native enzyme indicates that yeast acetyl-CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8. Several acyl-CoA derivatives of varying chain length were tested as substrates for yeast acetyl-CoA hydrolase. Although acetyl-CoA hydrolase was relatively specific for acetyl-CoA, longer acyl-chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl-CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine-modifying reagent.
乙酰辅酶A水解酶可将乙酰辅酶A水解为乙酸盐和辅酶A，该酶从酿酒酵母中分离得到，通过蛋白质序列分析表明其氨基末端被封闭。通过使用DEAE-琼脂糖、凝胶过滤和羟基磷灰石的连续纯化步骤，该酶被纯化了1080倍，达到表观均一性。通过凝胶过滤色谱法估计天然酵母乙酰辅酶A水解酶的分子量为64±5 kDa。SDS/PAGE分析显示变性分子量为65±2 kDa，结合天然酶的分子量表明酵母乙酰辅酶A水解酶是单体。该酶的最适pH接近8.0，其pI约为5.8。测试了几种不同链长的酰基辅酶A衍生物作为酵母乙酰辅酶A水解酶的底物。尽管乙酰辅酶A水解酶对乙酰辅酶A具有相对特异性，但较长链的酰基辅酶A也能被水解，并且在乙酰辅酶A水解过程中能够作为抑制剂发挥作用。在一系列二价阳离子中，Zn2+被证明是最有效的抑制剂。该酶被组氨酸修饰试剂焦碳酸二乙酯化学修饰而失活。
