Buttlaire D H, Chon M
J Biol Chem. 1977 Mar 25;252(6):1957-64.
The interactions of substrates with succinyl-CoA synthetase were investigated by measuring the enhancement of the longitudinal water proton relaxation rate (PRR) due to Mn(II) to the enzyme substrate complexes. The binding of Mn(II) to the enzyme was investigated by EPR. The effects of phosphorylating the enzyme on its interactions with Mn(II) and substrates were also examined. Mn(II) binds weakly to dephosphosuccinyl-CoA synthetase (E) at approximately four sites with a KD value of 0.14 mM, and the PRR enhancement of the complex, epsilonb, at 24.3 MHZ and 25 degree is 18.8. The phosphoenzyme (E-P) binds Mn(II) more strongly at approximately four sites with a KD value of 0.74 mM, and only a small change in epsilonb to 18.1. Mm ADP binds to E at one or two sites with K2 = 0.5 muM, the values of epsilont for the ternary E-Mn-ADP complex is 17.0. Free ADP binds about 126 times more weakly to the enzyme than does Mn-ADP. PRR titrations indicated that the values of epsilont for the ternary E-Mn-ADP and (E-P)-Mn-ADP complexes are about the same. Mn-ATP binds very weakly or not at all to (E-P)-Mn. Formation of the ternary complexes of CoA with E-Mn or (E-P)-Mn could be followed by small but significant increases in the PRR enhancement. No ternary complex with succinate could be detected since the addition of succinate had no effect on the PRR enhancement. However, a large decrease in enhancement, at least 2-fold, was observed upon addition of both succinate and CoA. An increase in the PRR enhancement was produced by the interaction of succinyl-CoA with the E-Mn complex. Upper limits of the dissociation constants for CoA from the quaternary E-Mn-ADP-succinate-CoA complex and for succinyl-CoA from the quaternary E-Mn-ADP-succinyl-CoA complex are 390 and 560 muM, respectively. The epsilon values for the quaternary and quinary complexes are 6.4 and 3.1, respectively. The successive occupation of substrate binding sites of succinyl-CoA synthetase produces alterations in the molecular dynamics or in the conformation of the active site (or both), which are accompanied by progressive decreases in the values of epsilon. Thus, the physical parameter used in these studies relects the previously observed catalytic properties of the enzyme system inasmuch as the catalytic function of succinyl-CoA synthetase is potentiated by substrate binding, and catalytic avtivity in partial reactions is maximized as binding sites are successively occupied.
通过测量由于锰（II）与酶 - 底物复合物导致的纵向水质子弛豫率（PRR）增强，研究了底物与琥珀酰辅酶A合成酶的相互作用。通过电子顺磁共振（EPR）研究了锰（II）与该酶的结合。还研究了酶磷酸化对其与锰（II）和底物相互作用的影响。锰（II）以约0.14 mM的KD值在大约四个位点与去磷酸化琥珀酰辅酶A合成酶（E）弱结合，并且在24.3 MHz和25℃下复合物的PRR增强值εb为18.8。磷酸化酶（E - P）以约0.74 mM的KD值在大约四个位点更强烈地结合锰（II），并且εb仅略有变化至18.1。毫米ADP以K2 = 0.5 μM在一个或两个位点与E结合，三元E - Mn - ADP复合物的εt值为17.0。游离ADP与该酶的结合比Mn - ADP弱约126倍。PRR滴定表明三元E - Mn - ADP和（E - P） - Mn - ADP复合物的εt值大致相同。Mn - ATP与（E - P） - Mn结合非常弱或根本不结合。辅酶A与E - Mn或（E - P） - Mn形成三元复合物后，PRR增强会有小但显著的增加。由于添加琥珀酸对PRR增强没有影响，因此未检测到与琥珀酸的三元复合物。然而，添加琥珀酸和辅酶A后观察到增强至少降低了2倍。琥珀酰辅酶A与E - Mn复合物的相互作用导致PRR增强增加。辅酶A从四元E - Mn - ADP - 琥珀酸 - 辅酶A复合物和解离常数的上限以及琥珀酰辅酶A从四元E - Mn - ADP - 琥珀酰辅酶A复合物的解离常数上限分别为390和560 μM。四元和五元复合物的ε值分别为6.4和3.1。琥珀酰辅酶A合成酶底物结合位点的连续占据会导致分子动力学或活性位点构象（或两者）的改变，这伴随着ε值的逐渐降低。因此，这些研究中使用的物理参数反映了先前观察到的酶系统的催化特性，因为琥珀酰辅酶A合成酶的催化功能通过底物结合得到增强，并且随着结合位点被相继占据，部分反应中的催化活性最大化。
