a+Ketoglutarate Dehydrogenase Complex of Acetobacter xylinum

The cu-ketoglutarate dehydrogenase complex of Acetobacterxylinum was purified to homogeneity. It consists of three main polypeptide chains with a total molecular weight of about 2.4 x 106. It catalyzes the overall Mg*+ and thiamin pyrophosphate-dependent, NAD+and CoA-linked oxidative decarboxylation of cY-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 a-ketoglutarate (Hill coefficient (n,,) = 2.0; concentration of ligand at half-maximum effect (S,,,) = 8 mM). The sigmoidal [cY-ketoglutaratel-velocity relationship became hyperbolic upon addition of AMP or Y-acetylpyridine adenine dinucleotide (AcPyAD) or in the presence of high concentrations of NAD. S,,, (Ir+ietoglutarate) decreased to 1 mM, but V,,,,, was unchanged. Saturation cuwes for NAD and AMP are sigmoidal (n, = 2) at low u+ketoglutarate concentrations and become hyperbolic at high a-ketoglutarate concentrations. As judged by S,,,, the relative efficiency of the allosteric effecters is AcPyAD>AMP>cu-ketoglutarate>NAD+. Half-maximal changes in nH, ,!&, and activation by AMP occur at a pH significantly different from that of halfmaximal activity. A model for the allosteric behavior of the complex is proposed in which the first enzyme component of the complex (E,) 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 (K, = 20 FM) and by succinyl-CoA with respect of CoA (K, = 3 PM). The properties of the cu-ketoglutarate dehydrogenase complex of A. xylinum appear to provide for appropriate partitioning of a+ketoglutarate carbon between competing pathways in response to the energy state of the cells.