Regulation of the 2-oxoglutarate dehydrogenase lipoate succinyltransferase complex from cauliflower by nucleotide. Pre-steady state kinetics and physical studies.

Analysis of the binding of thiamin pyrophosphate to the 2-oxoglutarate dehydrogenaselipoate succinyltransferase multienzyme complex using pre-steady state kinetic methods revealed that the presence of 2-oxoglutarate is not necessary for binding, although it does stabilize the complex by slowing the rate of dissociation of the holoenzyme. The rate of binding of thiamin-PPi to the enzyme and the subsequent enzyme activation are not limited by a reaction at C-2 of the thiazolium ring of thiamin-PPi since no kinetic isotope effect is observed when 2-D-thiamin-PPi is substituted for the protonated cofactor. The presence of 5'-AMP, which activates the reaction producing both a V and a Km response, causes a significant increase in kon for thiamin-PPi. The AMP analog 1,N6-ethenoadenosine-5'-monophosphate (epsilon-AMP) also activates the reaction, but shows only a K effect, with no influence on V. This effector reduces Kd for the thiamin-PPi2-oxoglutarate dehydrogenase complex by increasing kon. The change in kon for thiamin-PPi in response to changes in hydrogen ion concentration shows pK values which are unaffected by the addition of AMP, in this respect resembling the steady state kinetic response of V/Km and differing from the pH profile of V. The dissociation constant of holoenzyme is relatively insensitive to pH over the range pH 6 to 9, but in the presence of AMP the Kd, which is decreased in the range from pH 7 to 8, increases sharply at higher or lower pH values.