Ray W J, Post C B, Puvathingal J M
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Biochemistry. 1989 Jan 24;28(2):559-69. doi: 10.1021/bi00428a022.
Net rate constants that define the steady-state rate through a sequence of steps and the corresponding effective energy barriers for two (PO3-)-transfer steps in the phosphoglucomutase reaction were compared as a function of metal ion, M, where M = Mg2+ and Cd2+. These steps involve the reaction of either the 1-phosphate or the 6-phosphate of glucose 1,6-bisphosphate (Glc-P2) bound to the dephosphoenzyme (ED) to produce the phosphoenzyme (EP) and the free monophosphates, glucose 1-phosphate (Glc-1-P) or glucose 6-phosphate (Glc-6-P): EP.M + Glc-1-P----ED.M.Glc-P2----EP.M.Glc-6-P6. Before this comparison was made, net rate constants for the Cd2+ enzyme, obtained at high enzyme concentration via 31P NMR saturation-transfer studies [Post, C. B., Ray, W. J., Jr., & Gorenstein, D. G. (1989) Biochemistry (preceding paper in this issue)], were appropriately scaled by using the observed constants to calculate both the expected isotope-transfer rate at equilibrium and the steady-state rate under initial velocity conditions and comparing the calculated values with those measured in dilute solution. For the Mg2+ enzyme, narrow limits on possible values of the corresponding net rate constants were imposed on the basis of initial velocity rate constants for the forward and reverse directions plus values for the equilibrium distribution of central complexes, since direct measurement is not feasible. The effective energy barriers for both the Mg2+ and Cd2+ enzymes, calculated from the respective net rate constants, together with previously values for the equilibrium distribution of complexes in both enzymic systems [Ray, W. J., Jr., & Long, J. W. (1976) Biochemistry 15, 4018-4025], show that the 100-fold decrease in the kappa cat for the Cd2+ relative to the Mg2+ enzyme is caused by two factors: the increased stability of the intermediate bisphosphate complex and the decreased ability to cope with the phosphate ester involving the 1-hydroxyl group of the glucose ring. In fact, it is unlikely that the efficiency of (PO3-) transfer to the 6-hydroxyl group of bound Glc-1-P (thermodynamically favorable direction) is reduced by more than an order of magnitude in the Cd2+ enzyme. By contrast, the efficiency of the Li+ enzyme in the same (PO3-)-transfer step is less than 4 x 10(-8) that of the Mg2+ enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
比较了定义通过一系列步骤的稳态速率的净速率常数，以及磷酸葡萄糖变位酶反应中两个（PO₃⁻）转移步骤对应的有效能垒，该比较是作为金属离子M的函数进行的，其中M = Mg²⁺ 和Cd²⁺。这些步骤涉及与脱磷酸酶（ED）结合的葡萄糖1,6 - 二磷酸（Glc - P₂）的1 - 磷酸或6 - 磷酸发生反应，生成磷酸酶（EP）和游离单磷酸，即葡萄糖1 - 磷酸（Glc - 1 - P）或葡萄糖6 - 磷酸（Glc - 6 - P）：EP.M + Glc - 1 - P----ED.M.Glc - P₂----EP.M.Glc - 6 - P₆。在进行此比较之前，通过³¹P NMR饱和转移研究[Post, C. B., Ray, W. J., Jr., & Gorenstein, D. G. (1989) Biochemistry（本期之前的论文）]在高酶浓度下获得的Cd²⁺ 酶的净速率常数，通过使用观察到的常数进行适当缩放，以计算平衡时预期的同位素转移速率和初始速度条件下的稳态速率，并将计算值与在稀溶液中测量的值进行比较。对于Mg²⁺ 酶，由于直接测量不可行，基于正向和反向的初始速度速率常数以及中心复合物平衡分布的值，对相应净速率常数的可能值施加了狭窄的限制。根据各自的净速率常数计算出的Mg²⁺ 和Cd²⁺ 酶的有效能垒，连同之前两个酶系统中复合物平衡分布的值[Ray, W. J., Jr., & Long, J. W. (1976) Biochemistry 15, 4018 - 4025]表明，相对于Mg²⁺ 酶，Cd²⁺ 酶的催化常数κcat下降100倍是由两个因素引起的：中间双磷酸复合物稳定性的增加以及应对涉及葡萄糖环1 - 羟基的磷酸酯的能力下降。实际上，在Cd²⁺ 酶中，（PO₃⁻）转移到结合的Glc - 1 - P的6 - 羟基（热力学有利方向）的效率降低不太可能超过一个数量级。相比之下，Li⁺ 酶在相同（PO₃⁻）转移步骤中的效率不到Mg²⁺ 酶的4×10⁻⁸。（摘要截断于400字）
