Griffith A D, Cyr D M, Egan S G, Tremblay G C
Department of Biochemistry and Biophysics, University of Rhode Island, Kingston 02881.
Arch Biochem Biophys. 1989 Feb 15;269(1):201-7. doi: 10.1016/0003-9861(89)90101-x.
Pyruvate-dependent CO2 fixation by isolated mitochondria was strongly inhibited by sodium benzoate. Pyruvate carboxylase was identified as a site of inhibition by limiting flux measurements to assays of pyruvate carboxylase coupled with malate dehydrogenase. Benzoate reduced pyruvate-dependent incorporation of [14C]KHCO3 into malate and pyruvate-dependent malate accumulation by 74 and 72%, respectively. Aspartate-dependent malate accumulation was insensitive to benzoate, ruling out malate dehydrogenase as a site of action. Inhibition by benzoate was antagonized by glycine, which sharply accelerated conversion of benzoate to hippurate. Assays of coenzyme A and its acyl derivatives revealed inhibition to correlate with depletion of acetyl CoA and accumulation of benzoyl CoA. Depletion of acetyl CoA was sufficient to account for greater than 50% reduction in pyruvate carboxylase activity. Competition between acetyl CoA and benzoyl CoA for the activator site on pyruvate carboxylase was insignificant. Results support the interpretation that the observed inhibition of pyruvate carboxylase occurred primarily by depletion of the activator, acetyl CoA, through sequestration of coenzyme A during benzoate metabolism.
苯甲酸钠强烈抑制分离的线粒体中丙酮酸依赖性的二氧化碳固定。通过将通量测量限制在丙酮酸羧化酶与苹果酸脱氢酶偶联的测定中，确定丙酮酸羧化酶是抑制位点。苯甲酸盐分别使丙酮酸依赖性的[14C]KHCO3掺入苹果酸和丙酮酸依赖性的苹果酸积累减少74%和72%。天冬氨酸依赖性的苹果酸积累对苯甲酸盐不敏感，排除了苹果酸脱氢酶作为作用位点。甘氨酸可拮抗苯甲酸盐的抑制作用，甘氨酸能大幅加速苯甲酸盐向马尿酸盐的转化。辅酶A及其酰基衍生物的测定表明，抑制作用与乙酰辅酶A的消耗和苯甲酰辅酶A的积累相关。乙酰辅酶A的消耗足以解释丙酮酸羧化酶活性降低超过50%的原因。乙酰辅酶A和苯甲酰辅酶A在丙酮酸羧化酶激活位点上的竞争不显著。结果支持这样的解释，即观察到的丙酮酸羧化酶抑制主要是由于在苯甲酸盐代谢过程中辅酶A被隔离，从而使激活剂乙酰辅酶A耗尽所致。
