木醋杆菌的α-酮戊二酸脱氢酶复合体。纯化及调控特性。

Alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum. Purification and regulatory properties.

作者信息

Kornfeld S, Benziman M, Milner Y

出版信息

J Biol Chem. 1977 May 10;252(9):2940-7.

Abstract

The alpha-ketoglutarate dehydrogenase complex of Acetobacter xylinum was purified to homogeneity. It consists of three main polypeptide chains with a total molecular weight of about 2.4 X 10(6). It catalyzes the overall Mg2+ and thiamin pyrophosphate-dependent, NAD+- and CoA-linked oxidative decarboxylation of alpha-ketoglutarate, as well as the partial reactions characteristic of the three enzyme components described for the complex from other sources. Initial velocity studies revealed marked positive cooperativity for the substrate alpha-ketoglutarate (Hill coefficient (nH) = 2.0; concentration of ligand at half-maximum effect (S0.5) = 8 mM). The sigmoidal [alpha-ketoglutarate]-velocity relationship became hyperbolic upon addition of AMP or 3-acetylpyridine adenine dinucleotide (AcPyAD) or in the presence of high concentrations of NAD. S0.5 (alpha-ketoglutarate) decreased to 1 mM, but Vmax was unchanged. Saturation curves for NAD and AMP are sigmoidal (nH = 2) at low alpha-ketoglutarate concentrations and become hyperbolic at high alpha-ketoglutarate concentrations. As judged by S0.5, the relative efficiency of the allosteric effectors is AcPyAD greater than AMP greater than alpha-ketoglutarate- greater than NAD+. Half-maximal changes in nH, S0.5, and activation by AMP occur at a pH significantly different from that of half-maximal activity. A model for the allosteric behavior of the complex is proposed in which the first enzyme component of the complex (E1) is the site for the allosteric interactions and AMP is the primary positive modifier, whereas NAD and AcPyAD act as AMP analogues. The overall reaction is competitively inhibited by NADH with respect to NAD (K1 = 20 micronM) and by succinyl-CoA with respect of CoA (K1 = 3 micronM). The properties of the alpha-ketoglutarate dehydrogenase complex of A. xylinum appear to provide for appropriate partitioning of alpha-ketoglutarate carbon between competing pathways in response to the energy state of the cells.

摘要

木醋杆菌的α-酮戊二酸脱氢酶复合体被纯化至同质。它由三条主要多肽链组成，总分子量约为2.4×10⁶。它催化α-酮戊二酸的整体Mg²⁺和硫胺素焦磷酸依赖性、NAD⁺和CoA连接的氧化脱羧反应，以及从其他来源描述的该复合体的三种酶成分的部分反应。初始速度研究表明，底物α-酮戊二酸具有显著的正协同性（希尔系数（nH）=2.0；半最大效应时的配体浓度（S0.5）=8 mM）。加入AMP或3-乙酰吡啶腺嘌呤二核苷酸（AcPyAD）后，或在高浓度NAD存在下，S形的[α-酮戊二酸]-速度关系变为双曲线。S0.5（α-酮戊二酸）降至1 mM，但Vmax不变。在低α-酮戊二酸浓度下，NAD和AMP的饱和曲线为S形（nH = 2），在高α-酮戊二酸浓度下变为双曲线。根据S0.5判断，变构效应剂的相对效率为AcPyAD＞AMP＞α-酮戊二酸＞NAD⁺。nH、S0.5和AMP激活的半最大变化发生在与半最大活性显著不同的pH值下。提出了该复合体变构行为的模型，其中复合体的第一个酶成分（E1）是变构相互作用的位点，AMP是主要的正调节剂，而NAD和AcPyAD作为AMP类似物起作用。整体反应相对于NAD被NADH竞争性抑制（K1 = 20 μM），相对于CoA被琥珀酰辅酶A竞争性抑制（K1 = 3 μM）。木醋杆菌的α-酮戊二酸脱氢酶复合体的特性似乎能根据细胞的能量状态，在竞争途径之间适当分配α-酮戊二酸碳。