PURIFICATION AND CHARACTERIZATION OF ACETOACETYL-COA SYNTHETASE FROM ZOOGLOEA-RAMIGERA I-16-M
PURIFICATION AND CHARACTERIZATION OF ACETOACETYL-COA SYNTHETASE FROM ZOOGLOEA-RAMIGERA I-16-M
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DOI：
10.1111/j.1432-1033.1982.tb06889.x
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发表时间：
1982-01-01
期刊：
影响因子：
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通讯作者：
TOMITA, K
中科院分区：
文献类型：
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作者：
FUKUI, T;ITO, M;TOMITA, K
从缺乏3-酮酸辅酶A转移酶的聚(3-羟基丁酸酯)聚合菌Z.ramigera I-16-M中分离纯化了乙酰乙酰-辅酶A合成酶。纯化后的酶比活力为52.2微克分子乙酰乙酰-辅酶A形成的min~(-1)mg蛋白~(-1)，比粗提物纯化680倍，产率51%。该酶完全需要ATP、CoA一价阳离子(K+、Rb+、Cs+或NH4+)和二价阳离子(Mg2+、Mn2+、Ca2+或Ni2+)来激活乙酰乙酸酯，生成等摩尔量的乙酰乙酰CoA、AMP和PPI。该酶反应的最适pH为8.4。该酶的相对分子质量为.apprx。在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳法估计为70,000，Sephadex G-200凝胶过滤估计为72,000。对乙酰乙酸酯、L(+)-3-羟基丁酸酯、三磷酸腺苷和辅酶A的Km值为7.6倍。10-5M，1.4倍。10-3M，3.3倍。10-5M和9.1倍。10~(-5)M。
Acetoacetyl-CoA synthetase was purified to electrophoretic homogeneity from Z. ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium, which lacks 3-ketoacid CoA-transferase. The purified enzyme has a specific activity of 52.2 .mu.mol acetoacetyl-CoA formed min-1 mg protein-1, which constituted a 680-fold purification compared to the crude extract, with a 51% yield. The enzyme absolutely required ATP, CoA a monovalent cation (K+, Rb+, Cs+ or NH4+) and a divalent cation (Mg2+, Mn2+, Ca2+ or Ni2+) for the activation of acetoacetate, yielding acetoacetyl-CoA, AMP and PPi in equimolar amounts. The pH optimum of the enzyme reaction was 8.4. The MW of the enzyme was .apprx. 70,000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and 72,000 by Sephadex G-200 gel filtration. The enzyme was active only on acetoacetate and to a lesser extent on L(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L(+)-3-hydroxybutyrate, ATP and CoA were 7.6 .times. 10-5 M, 1.4 .times. 10-3 M, 3.3 .times. 10-5 M and 9.1 .times. 10-5 M, respectively.