Andi Babak, West Ann H, Cook Paul F
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
J Biol Chem. 2005 Sep 9;280(36):31624-32. doi: 10.1074/jbc.M502846200. Epub 2005 May 16.
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.
高柠檬酸合酶（HCS）催化真菌中赖氨酸生物合成的α-氨基己二酸途径的一个受调控步骤。通过测量有无效应物存在时的初始速率，研究了酿酒酵母中HCS受Na⁺和反馈抑制剂赖氨酸调控的动力学机制。数据表明，低浓度时Na⁺是激活剂，高浓度时是抑制剂，这些效应是由于单价离子与游离酶中的两个不同位点结合所致。Na⁺的抑制和激活可同时发生，酶的净速率由Na⁺/K（iNa⁺）和Na⁺/K（act）决定，其中K（iNa⁺）和K（act）分别是抑制常数和激活常数。在高浓度的乙酰辅酶A（第二种结合底物）存在时，Na⁺的抑制作用消除，但激活作用仍然存在。荧光结合研究表明，赖氨酸作为抑制剂与其结合位点具有高亲和力结合。赖氨酸的抑制作用相对于α-酮戊二酸是竞争性的，在赖氨酸浓度高达5 mM的生理范围内呈线性关系。Na⁺和赖氨酸的作用相互独立。建立了一个考虑上述所有效应的HCS调控模型。
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