Johnson J K, Srivastava D K
Department of Biochemistry, North Dakota State University, Fargo 58105.
Arch Biochem Biophys. 1991 Jun;287(2):250-6. doi: 10.1016/0003-9861(91)90414-e.
The fluorescence polarization of 8-hydroxypyrene (1,3,6)trisulfonate (HPT) increases upon interaction with pig heart citrate synthase. Titration of HPT with increasing concentrations of citrate synthase exhibits a hyperbolic saturation behavior, from which the dissociation constant of the enzyme-HPT complex (3.64 +/- 0.3 microM) was determined. The enzyme-HPT interaction is competitively inhibited by oxaloacetate (but not affected by acetyl CoA) with a Ki of 4.3 +/- 1.8 microM. This value is similar to the dissociation constant (Kd = 4.5 +/- 1.6 microM) for the enzyme-oxalocetate complex (determined in the absence of any effector ligand), as well as to the Km for oxaloacetate (3.9 +/- 0.7 microM) in a steady-state citrate synthase catalyzed reaction at a saturating concentration of acetyl CoA. However, the dissociation constant for the citrate synthase-oxaloacetate complex determined by the urea denaturation method is at least 25-fold lower than those determined by the other methods. This suggests an effector role of urea in strengthening the enzyme-oxaloacetate interaction. At low nondenaturing concentrations, urea inhibits the citrate synthase catalyzed reaction in an uncompetitive manner with respect to oxaloacetate, i.e., the Km for oxaloacetate decreases with an increase in urea concentration. This further suggests that urea stabilizes the interaction between citrate synthase and oxaloacetate. The effect of urea is specific for the substrate oxaloacetate, and not for the substrate analogue, HPT, although both these ligands bind citrate synthase with equal affinities, and protect the enzyme against thermal denaturation with equal magnitudes. The results presented herein are discussed in the light of known conformational states of the enzyme.
8-羟基芘（1,3,6）三磺酸盐（HPT）与猪心柠檬酸合酶相互作用时，其荧光偏振度会增加。用浓度不断增加的柠檬酸合酶滴定HPT呈现出双曲线饱和行为，据此确定了酶-HPT复合物的解离常数（3.64±0.3微摩尔）。草酰乙酸对酶-HPT相互作用有竞争性抑制作用（但不受乙酰辅酶A影响），抑制常数Ki为4.3±1.8微摩尔。该值与酶-草酰乙酸复合物的解离常数（Kd = 4.5±1.6微摩尔，在无任何效应配体的情况下测定）以及在乙酰辅酶A饱和浓度下稳态柠檬酸合酶催化反应中草酰乙酸的米氏常数（3.9±0.7微摩尔）相似。然而，通过尿素变性法测定的柠檬酸合酶-草酰乙酸复合物的解离常数至少比其他方法测定的值低25倍。这表明尿素在增强酶-草酰乙酸相互作用方面具有效应作用。在低非变性浓度下，尿素对草酰乙酸以非竞争性方式抑制柠檬酸合酶催化的反应，即草酰乙酸的米氏常数随尿素浓度增加而降低。这进一步表明尿素稳定了柠檬酸合酶与草酰乙酸之间的相互作用。尿素的作用对底物草酰乙酸具有特异性，而对底物类似物HPT没有特异性，尽管这两种配体与柠檬酸合酶的结合亲和力相同，且对酶热变性的保护程度相同。本文根据该酶已知的构象状态对结果进行了讨论。
