Regulatory mechanism of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae. I. Kinetic studies.
Homocitrate synthase (HCS) catalyzes one of the regulated steps of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The kinetic mechanism of regulation of HCS from Saccharomyces cerevisiae by Na+ and the feedback inhibitor lysine was studied by measuring the initial rate in the absence and presence of the effectors. The data suggest that Na+ is an activator at low concentrations and an inhibitor at high concentrations and that these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na+ can occur simultaneously, with the net rate of the enzyme determined by Na+/K(iNa+) and Na+/K(act), where K(iNa+) and K(act) are the inhibition and activation constants, respectively. The inhibition by Na+ was eliminated at high concentrations of acetyl-CoA, the second substrate bound, but the activation remained. Fluorescence binding studies indicated that lysine bound with high affinity to its binding site as an inhibitor. The inhibition by lysine was competitive versus alpha-ketoglutarate and linear in the physiological range of lysine concentrations up to 5 mm. The effects of Na+ and lysine were independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above.
Andi
Babak
B
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
West
Ann H
AH
Cook
Paul F
PF
D000214:Acyl Coenzyme A / Q000378:metabolism; D002413:Cations, Divalent; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012964:Sodium / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000214:Acyl Coenzyme A; D002413:Cations, Divalent; D008024:Ligands; D012964:Sodium; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases; D008239:Lysine