Pickart C M, Jencks W P
J Biol Chem. 1984 Feb 10;259(3):1629-43.
A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.
基于在一组实验（公式；见正文）条件（pH 7.0、25℃、100 mM KCl、5 mM MgSO4）下方程1所示反应微观步骤的平衡常数，描述了肌浆网ATP酶催化钙转运的热力学循环：KCa = 5.9×10⁻¹² M²，KαATP = 15 μM，Kint = 0.47，KαADP = 0.73 mM，K'int = 1.7，K"Ca = 2.2×10⁻⁶ M²，Kp = 37 mM。K"Ca的值通过ATP水解自由能差值计算得出。Pi和E自发形成酰基磷酸酯是因为E-P中表达了12.5 kcal mol⁻¹的非共价结合能。E×Pi中仅表达了1.9 kcal mol⁻¹的结合能。在E-P×Ca²中，结合的磷酸根和钙相互不稳定，ΔGD = 7.6 kcal mol⁻¹，这使得磷酸根能够转移到ADP，钙能够转移到囊泡内的浓缩钙库中。有人提出，E-P×Ca²解离的有序动力学机制，即磷酸根在钙在外部解离之前转移到ADP，钙在内部解离之后磷酸根转移到水中，保留了这些配体的吉布斯自由能，并对转运过程中的偶联起主要作用。在pH 6下将E和Pi混合后，E-P出现之前的延迟（约5 ms）会被ATP和增加的[Pi]缩短。这表明ATP加速了Pi的结合。ATP在平衡时对E-P形成的微弱抑制也表明，在pH 6时ATP和磷酸根可以同时与酶结合。在pH 7下，从E-P、Ca²⁺和[³²P]ATP形成E-³²P×Ca²的反应序列中所有步骤的速率常数都大于或等于115 s⁻¹。E-P×Ca²以κ = 17 s⁻¹的速率分解，这表明它是一个动力学上可行的中间体。如果在2 mM Ca²⁺存在下形成中间体，κ的值会降至4 s⁻¹。这种降低以及大于0.1 mM Ca²⁺对周转的抑制可能是由于E-P×Ca³的缓慢分解导致的。
