Walters D W, Gilbert H F
J Biol Chem. 1986 Oct 5;261(28):13135-43.
Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.
鸡肝脂肪酸合酶在pH 7.2和8.0条件下，与低浓度的常见生物二硫化物（包括谷胱甘肽二硫化物、辅酶A二硫化物和谷胱甘肽 - 辅酶A混合二硫化物）孵育时会迅速失活并发生交联。该酶被谷胱甘肽二硫化物失活的同时，每个单体总共4 - 5个酶硫醇会被氧化。每个单体仅结合一个谷胱甘肽当量作为蛋白质混合二硫化物，其结合速率明显慢于失活速率。蛋白质 - SS - 蛋白质二硫化物的形成导致酶亚基显著交联。失活的酶可通过与包括二硫苏糖醇、巯基乙醇和谷胱甘肽在内的硫醇（10 - 20 mM）孵育而迅速且完全地重新激活，并且交联完全逆转。在谷胱甘肽氧化还原缓冲液（GSH + GSSG）中，二硫键形成达到平衡。平衡时的酶活性既取决于谷胱甘肽与谷胱甘肽二硫化物的比例，也取决于总谷胱甘肽浓度。脂肪酸合酶在谷胱甘肽氧化还原缓冲液中的氧化还原平衡常数为15 mM（Ered + GSSG ⇌ Eox + 2GSH）。每个单体至少形成一个蛋白质 - 蛋白质二硫化物主导了该酶的氧化还原特性，而与谷胱甘肽形成一个蛋白质混合二硫化物（Kmixed = 0.45）对活性影响很小。氧化平衡常数表明，响应肝脏谷胱甘肽状态可能的生理变化，还原型和氧化型酶之间不会有显著的循环。本文讨论了细胞谷胱甘肽浓度的变化可能作为其他酶代谢控制机制的可能性。
