羧化酶(TC)是一种含生物素的酶,其催化羧基从甲基丙二酰-CoA转移到丙酮酸以形成丙酰-CoA和草乙酸酯。转移是通过与多亚基酶复合物内的1.3S亚基结合的羧化生物素实现的。TC的1.3S亚基是一个123个氨基酸的多肽,生物素共价连接在Lys89上。我们已在大肠杆菌中过表达1.3S,并通过1D和2D-NMR光谱表征了生物素化和apo形式。为了寻找蛋白质-生物素的相互作用,可以调节1.3S亚基上生物素环的反应性,我们比较了化学变化,松弛参数,游离和1.3S结合生物素的脲基环质子的NH交换速率。这些性质对于两种形式的生物素是相似的。Further,对1.3S的NOE实验显示,生物素和蛋白质之间没有可检测到的交叉峰。与这些发现一致,holo-和apo-1.3S的2DNMR数据基本相同,表明两种形式的蛋白质之间的构象变化很小或没有变化。1.3S中不存在强蛋白质-生物素相互作用的结论与来自大肠杆菌乙酰辅酶A羧化酶的生物素羧化酶载体蛋白的发现相反,揭示了显著的生物素-蛋白质接触[Athappilly,F.K.,和亨德里克森,W.A.(1995)结构3,1407-1419]。Further,为1.3S确定的生物素NH1交换率表明,在TC(pH5.5-6.5)的最佳活性区域中,酸催化的交换占主导地位。在该pH范围内,碱催化的速率太小(<1s-1),不能说明酶的转换率。因此,对于甲基丙二酰辅酶A中的羧基的亲核攻击,N1'原子被激活的方式似乎并不仅仅依赖于1.3S亚单位内的相互作用;相反,激活必须发生在全酶中的亚单位的界面。
Transcarboxylase (TC) is a biotin-containing enzyme catalyzing the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. The transfer is achieved via carboxylated biotin bound to a 1.3S subunit within the multisubunit enzyme complex. The 1.3S subunit of TC is a 123 amino acid polypeptide, to which biotin is covalently attached at Lys 89. We have overexpressed 1.3S in Escherichia coli and characterized the biotinylated and apo-forms by 1D- and 2D-NMR spectroscopy. To search for protein-biotin interactions, which could modulate the reactivity of the biotin ring on the 1.3S subunit, we have compared the chemical shifts, relaxation parameters, and NH exchange rates of the ureido ring protons of free and 1.3S-bound biotin. These properties are similar for both forms of the biotin. Further, NOE experiments on 1.3S revealed no detectable cross peaks between biotin and the protein. Consistent with these findings, the 2D NMR data for holo- and apo-1.3S are essentially identical indicating little or no changes in conformation between the two forms of the protein. The conclusion that strong protein-biotin interactions do not exist in 1.3S contrasts with the findings for the biotin carboxylase carrier protein from E. coli acetyl-CoA carboxylase, which reveal significant biotin-protein contacts [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419]. Further, the biotin NH1\' exchange rates determined for 1.3S show that in the region of optimal activity for TC (pH 5.5-6.5) acid-catalyzed exchange predominates. In this pH range the base-catalyzed rate is too small (< 1 s-1) to account for the turnover rate of the enzyme. Thus, the means by which the N1\' atom is activated for nucleophilic attack of the carboxyl group in methylmalonyl-CoA does not appear to depend on interactions within the 1.3S subunit alone; rather activation must occur at the interfaces of the subunits in the holoenzyme.