Schramm V L, McCluskey R, Emig F A, Litwack G
J Biol Chem. 1984 Jan 25;259(2):714-22.
Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer YaYa of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 microM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl] glutathione (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 microM. Analysis of the glutathione S-transferase X sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the glutathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 microM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 microM, and 25 microM were obtained for glutathione, 1-Cl-2,4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl) glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.
通过对底物、产物以及谷胱甘肽的自旋标记产物类似物（sl-谷胱甘肽）进行动力学和结合研究，以表征谷胱甘肽S-转移酶同二聚体YaYa催化位点的动力学机制和特性。产物抑制研究以及sl-谷胱甘肽的抑制作用表明底物是随机添加的。通过动力学测定得出产物S-(2,4-二硝基苯基)谷胱甘肽的解离常数约为7微摩尔。一种新描述的自旋标记产物类似物，S-[[(2,2,5,5-四甲基-1-氧代-3-吡咯烷基)-氨基甲酰基]甲基]谷胱甘肽（sl-谷胱甘肽），对两种底物（谷胱甘肽和1-氯-2,4-二硝基苯）均表现为竞争性抑制剂，通过动力学测定其解离常数约为40微摩尔。利用电子顺磁共振（EPR）对谷胱甘肽S-转移酶X sl-谷胱甘肽复合物进行分析，得到一个刚性极限光谱，表明自旋标记高度固定。动力学和EPR结果支持以下提议：sl-谷胱甘肽通过占据谷胱甘肽和疏水底物位点，作为双底物或产物类似物进行结合。通过EPR对sl-谷胱甘肽进行结合研究，得出解离常数为28微摩尔，且每个同二聚体有一个单一结合位点。利用底物和产物对sl-谷胱甘肽的置换作用来直接测定酶-配体解离常数。当将酶加入到sl-谷胱甘肽和竞争性配体的混合物中时，谷胱甘肽、1-氯-2,4-二硝基苯和S-(2,4-二硝基苯基)谷胱甘肽的解离常数分别为2.1毫摩尔、17微摩尔和25微摩尔。谷胱甘肽和1-氯-2,4-二硝基苯的解离常数，但S-(2,4-二硝基苯基)谷胱甘肽的解离常数不依赖于添加顺序，这与该酶存在几种动力学稳定构象一致。本文所述的sl-谷胱甘肽可能为对其他谷胱甘肽S-转移酶或其他结合谷胱甘肽的酶进行类似研究提供一种有用的类似物。
