Overexpression and kinetic characterization of the carboxyltransferase component of acetyl-CoA carboxylase
Overexpression and kinetic characterization of the carboxyltransferase component of acetyl-CoA carboxylase
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DOI：
10.1074/jbc.273.30.19140
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发表时间：
1998-07-24
影响因子：
4.8
通讯作者：
Waldrop, GL
中科院分区：
文献类型：
--
作者：
Blanchard, CZ;Waldrop, GL
乙酰辅酶A羧基酶催化脂肪酸生物合成的第一步。该酶的大肠杆菌形式由生物素羧基酶蛋白、生物素羧基载体蛋白和羧基转移酶蛋白组成。在这份报告中，我们描述了羧基转移酶组分基因的过度表达。重组羧基转移酶的稳态动力学表现为相反方向，丙二酰辅酶A与生物细胞素反应生成乙酰辅酶A和羧基生物细胞素。初始速度图表明，动力学机制是平衡有序的，丙二酰辅酶A结合在生物细胞素之前，丙二酰辅酶A在平衡时与羧基转移酶结合。生物素类似物去硫生物素和2-咪唑酮对羧基转移酶有抑制作用。这两个类似物都表现出抛物线非竞争性抑制，这意味着两个抑制剂分子与酶结合。在低pH条件下，最大速率(V)和(V/K)(生物细胞素)参数对pH的依赖性降低。在(V/K)生物细胞素谱中pK为6.2或更低且在V谱中pK为7.5的酶上的单个电离基团必须去质子化才能催化。N-乙基马来酰亚胺可使羧基转移酶失活，而丙二酰辅酶A可防止其失活。这表明催化需要活性中心内或活性中心附近的硫醇。N-乙基马来酰亚胺对羧基转移酶的失活速率随pH的降低而降低，表明羟基的pK值为7.3。提出半胱氨酸的硫代离子起催化碱的作用来去除生物细胞素中的N1‘质子。
Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report the overexpression of the genes for the carboxyltransferase component is described. The steady-state kinetics of the recombinant carboxyltransferase are characterized in the reverse direction, in which malonyl-CoA reacts with biocytin to form acetyl-CoA and carboxybiocytin. The initial velocity patterns indicated that the kinetic mechanism is equilibrium-ordered with malonyl-CoA binding before biocytin and the binding of malonyl-CoA to carboxyltransferase at equilibrium. The biotin analogs, desthiobiotin and 2-imidazolidone, inhibited carboxyltransferase. Both analogs exhibited parabolic noncompetitive inhibition, which means that two molecules of inhibitor bind to the enzyme. The pH dependence for both the maximum velocity (V) and the (V/K)(biocytin) parameters decreased at low pH. A single ionizing group on the enzyme with a pK of 6.2 or lower in the (V/K)biocytin profile and 7.5 in the V profile must be unprotonated for catalysis. Carboxyltransferase was inactivated by N-ethylmaleimide, whereas malonyl-CoA protected against inactivation. This suggests that a thiol in or near the active site is needed for catalysis. The rate of inactivation of carboxyltransferase by N-ethylmaleimide decreased with decreasing pH and indicated that the pK of the sulfhydryl group had a pK value of 7.3. It is proposed that the thiolate ion of a cysteine acts as a catalytic base to remove the N1' proton of biocytin.