Blanchard C Z, Lee Y M, Frantom P A, Waldrop G L
Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge 70803-1806, USA.
Biochemistry. 1999 Mar 16;38(11):3393-400. doi: 10.1021/bi982660a.
Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain 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 a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.
乙酰辅酶A羧化酶催化长链脂肪酸生物合成中的首个关键步骤。该酶的大肠杆菌形式由生物素羧化酶蛋白、生物素羧基载体蛋白和羧基转移酶蛋白组成。在本报告中，描述了一种对生物素羧化酶组分进行定点诱变的系统。源自染色体基因的酶的野生型拷贝与编码在质粒上的酶的突变形式分离。通过连接到酶突变形式氨基末端的组氨酸标签和镍亲和色谱来实现两种形式的分离。该系统用于将四个活性位点残基E211、E288、N290和R292突变为丙氨酸，随后针对生物素羧化酶催化的几种不同反应对其进行表征。与野生型生物素羧化酶相比，所有四种突变酶在所有不同测定中都给出了非常相似的结果，表明突变的残基具有共同功能。这些突变不影响依赖于碳酸氢盐的ATP酶反应。相反，这些突变使依赖于生物素的ATP酶反应的最大速度降低了1000倍，但不影响生物素的Km值。生物素羧化酶催化的氨甲酰磷酸与ADP反应的ATP合成反应活性因突变而降低了100倍。ATP合成反应在野生型中需要生物素来刺激活性；然而，生物素不刺激突变酶的活性。结果表明，这些突变消除了生物素增加酶活性的能力。因此，E211、E288、N290和R292至少部分负责生物素在生物素羧化酶中诱导的底物协同作用。
