Kinetic mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae.
Kinetic data have been collected suggesting a preferred sequential ordered kinetic mechanism for the histidine-tagged homocitrate synthase (HCS) from Saccharomyces cerevisiae with alpha-ketoglutarate binding before AcCoA and CoA released before homocitrate. Oxaloacetate is also a substrate for HCS, but with lower affinity than alpha-ketoglutarate. In agreement with the ordered kinetic mechanism desulfo-CoA is uncompetitive and citrate is competitive vs alpha-ketoglutarate. Varying AcCoA, citrate is a noncompetitive inhibitor as predicted, but CoA is noncompetitive vs AcCoA suggesting binding of CoA to E:homocitrate and E:alpha-ketoglutarate. The product CoA behaves in a manner identical to the dead-end analogue desulfo-CoA, suggesting an E:alpha-ketoglutarate:CoA dead-end complex. Data further suggest an irreversible reaction overall, in agreement with the downhill nature of the reaction as a result of homocitryl-CoA hydrolysis. Fluorescence titration data generally agree with the steady state data, but show finite binding of CoA and AcCoA to free enzyme, suggesting that the mechanism may be random with a high degree of synergism of binding between the reactants.
Andi
Babak
B
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
West
Ann H
AH
Cook
Paul F
PF
D000105:Acetyl Coenzyme A / Q000378:metabolism; D003505:Cyclodextrins / Q000378:metabolism; D008024:Ligands; D008433:Mathematics; D010071:Oxaloacetates / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000493:pharmacokinetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000493:pharmacokinetics*; D047391:alpha-Cyclodextrins
D003505:Cyclodextrins; D008024:Ligands; D010071:Oxaloacetates; D029701:Saccharomyces cerevisiae Proteins; D047391:alpha-Cyclodextrins; D000105:Acetyl Coenzyme A; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases; C032613:alpha-cyclodextrin