Stephan S, Hasselbach W
Max-Planck-Institut für Medizinische Forschung, Abteilung Physiologie, Heidelberg, Federal Republic of Germany.
Eur J Biochem. 1991 Dec 5;202(2):551-7. doi: 10.1111/j.1432-1033.1991.tb16407.x.
The effect which hydrostatic pressure exerts on the hydrolysis of dinitrophenyl phosphate and nitrophenyl phosphate by the sarcoplasmic reticulum calcium-transport enzyme was determined. Activation volumes for substrate hydrolysis at saturating and non-saturating concentrations of calcium were determined and used to evaluate volume increments for initial calcium binding. A reaction scheme in which two unidirectional substrate-driven reactions transfer high-affinity into low-affinity calcium-binding sites was applied to determine binding-volume increments. It has been inferred from the pressure dependence of the volume-generating function, defined as the difference between the reciprocal reaction rates of the saturated and the unsaturated enzyme, that calcium binding proceeds in two steps. The two associated binding constants are endowed with large binding-volume increments of opposite signs (+84 to +207 ml/mol and -3 to -136 ml/mol). Under different experimental conditions, with respect to the temperature, degree of calcium saturation and absence or presence of Me2SO, they add up to the same integral volume increment of 73 +/- 3.5 ml/mol for the entry of two calcium ions into the reaction cycle. In aqueous media, the two binding constants contribute about equally to binding and to the observed binding-volume increment. The presence of Me2SO strongly favours the first binding step. The size of the integral volume increment is in line with that determined for the interaction of calcium with calmodulin [Kupke, D.W. & Dorrier, T.E. (1986) Biochem. Biophys. Res. Commun. 38, 199-204].
测定了流体静压对肌浆网钙转运酶水解二硝基苯磷酸酯和硝基苯磷酸酯的影响。测定了在钙的饱和浓度和非饱和浓度下底物水解的活化体积，并用于评估初始钙结合的体积增量。应用一个反应方案来确定结合体积增量，在该方案中，两个单向底物驱动反应将高亲和力钙结合位点转化为低亲和力钙结合位点。根据定义为饱和酶和不饱和酶的倒数反应速率之差的体积生成函数的压力依赖性推断，钙结合分两步进行。两个相关的结合常数具有相反符号的大结合体积增量（+84至+207毫升/摩尔和-3至-136毫升/摩尔）。在不同的实验条件下，关于温度、钙饱和度以及是否存在二甲基亚砜（Me2SO），两个钙离子进入反应循环的总体积增量相同，为73±3.5毫升/摩尔。在水性介质中，两个结合常数对结合以及观察到的结合体积增量的贡献大致相等。二甲基亚砜的存在强烈有利于第一步结合。总体积增量的大小与钙与钙调蛋白相互作用所确定的大小一致[库普克，D.W. & 多里尔，T.E.（1986年）《生物化学与生物物理研究通讯》38卷，第199 - 204页]。
