Walters D W, Gilbert H F
J Biol Chem. 1986 Nov 25;261(33):15372-7.
Reversible thiol/disulfide exchange equilibria between rabbit muscle phosphofructokinase and glutathione redox buffers results in a dependence of the activity of the enzyme on the thiol to disulfide ratio of the redox buffer (Gilbert, H. F. (1982) J. Biol. Chem. 257, 12086-12091). The transition between fully reduced (active) and fully oxidized (inactive) enzyme is half complete at a [GSH]/[GSSG] ratio of 6.5 +/- 1 at pH 8.0 and 5.6 +/- 0.9 at pH 7.2. In the presence of excess GSSG approximately 40-50% of the activity is lost in a rapid process (k = 110 M-1 min-1), while the remaining activity is lost more slowly (k = 1.9 M-1 min-1). Two equivalents of radiolabeled glutathione are incorporated covalently, one coincident with each phase of inactivation. The most rapidly oxidized sulfhydryl group is also the most rapidly reduced by GSH in the reverse reaction (k = 150 M-1 min-1). Reduction of a more slowly reacting protein-glutathione mixed disulfide is required to regenerate the original activity (k = 0.33 M-1 min-1). The thiol/disulfide oxidation equilibrium constant (Kox) for the most rapidly oxidized sulfhydryl group is estimated to be 0.7 while that for the more slowly oxidized group is 6.1. The sulfhydryl group which is more easily oxidized kinetically is the more thermodynamically resistant to oxidation. The magnitude of the equilibrium constants for these reversible oxidations would suggest that the oxidation state (and activity) of phosphofructokinase would not be significantly affected by typical metabolic changes in the glutathione oxidation state in vivo.
兔肌肉磷酸果糖激酶与谷胱甘肽氧化还原缓冲液之间存在可逆的硫醇/二硫键交换平衡，这导致该酶的活性依赖于氧化还原缓冲液的硫醇与二硫键比例（吉尔伯特，H.F.（1982年）《生物化学杂志》257卷，12086 - 12091页）。在pH 8.0时，[GSH]/[GSSG]比例为6.5±1，在pH 7.2时为5.6±0.9时，完全还原（有活性）和完全氧化（无活性）的酶之间的转变完成一半。在过量GSSG存在下，约40 - 50%的活性在一个快速过程中丧失（k = 110 M⁻¹ min⁻¹），而剩余活性丧失得更慢（k = 1.9 M⁻¹ min⁻¹）。两当量的放射性标记谷胱甘肽共价结合，一个与失活的每个阶段同时发生。在反向反应中，氧化最快的巯基也被GSH还原得最快（k = 150 M⁻¹ min⁻¹）。需要还原反应较慢的蛋白质 - 谷胱甘肽混合二硫键才能恢复原始活性（k = 0.33 M⁻¹ min⁻¹）。氧化最快的巯基的硫醇/二硫键氧化平衡常数（Kox）估计为0.7，而氧化较慢的基团的Kox为6.1。在动力学上更容易氧化的巯基在热力学上更抗氧化。这些可逆氧化的平衡常数大小表明，体内谷胱甘肽氧化态的典型代谢变化不会显著影响磷酸果糖激酶的氧化态（和活性）。
