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.
D000217:Acyltransferases / Q000378:metabolism*; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007655:Ketoglutarate Dehydrogenase Complex / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013386:Succinates / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000494:pharmacology; D008063:Thioctic Acid / Q000378:metabolism
D009097:Multienzyme Complexes; D013386:Succinates; D008063:Thioctic Acid; D007658:Ketone Oxidoreductases; D007655:Ketoglutarate Dehydrogenase Complex; D000217:Acyltransferases; C021515:dihydrolipoamide succinyltransferase; D013835:Thiamine Pyrophosphate