Regulatory Properties of the a-Ketoglutarate Dehydrogenase Complex of Acetobacter xylinum

The initial velocity studies on the a-ketoglutarate dehydrogenase complex in toluene/Triton X-lOO-permeabilized Acetobwter xylinum cells revealed that the regulatory properties of the complex, in situ, are the same as for the purified complex. Thus, sigmoidal kinetics with respect to a-ketoglutarate were obtained (So.5 (a-ketogkltarate) = 5 lfl~ ; nH = 1.8), which convert into a hyperbolic relationship (SW (a-~etogl,,tarPte) = 0.8 m ; nH = 1.0) upon the addition of the allosteric effecters AMP, NAD, or 3-acetylpyridine adenine dinucleotide (AcPSTAD). The initial velocity studies of the El and Ea components of the multienzyme complex resulted in linear Michaelian patterns without effect by cr-ketoglutarate AMP or AcPyAD on the velocity. On the other hand, the EI component assayed within the complex by ferricyanide-linked a-ketoglutarate oxidation showed a typical sigmoidal velocity-a-ketoglutarate relationship (SO.5 (a-ketagl”urat.2) = 5 mu; nH = 2.0) which was converted into a hyperbolic relationship (SO.S (,,-ketoglutarate) = 1 mu; nH = 1.0) upon addition of AMP, NAD, or AcPyAD. A pK, = 8.2 of an unknown group for AMP activation was determined from the activation versus pH profile of the El reaction, in good agreement with the finding for the overall reaction. This sensitivity towards higher pH values was found not to be connected to the association of E1 or other components of the complex, as shown by gel filtration and ultracentrifugation at pH 6.8 and 8.4. In agreement with the Koshland model (Koshland, D. E., Jr., NBmethy, G., and Filmer, D. (1966) Biochemistry 5, 365-385) for allosteric protein interactions, the existence of multiple binding sites for M$+ l thiamin-PP in the El component was proved by using M&+0 thiaminPP as a specific cofactor. This was found both from steady state kinetics (at least four sites were evident) and from fluorometric titration of the complex (up to 3.2 mol of M&+=thiamin-PP bound/m01 of enzyme) using the specific fluorescence quenching phenomenon induced by M$+. thiamin-PP at 330 nm maximum emission. M&+ . thiamin-PP-induced fluorescence quenching was not affected by AMP or cr-ketoglutarate.