Petersen R L, Gupta B K
Biophys J. 1979 Jul;27(1):1-14. doi: 10.1016/S0006-3495(79)85198-X.
The interaction of CrADP, an exchange-inert paramagnetic analogue of Mg-ADP, with yeast hexokinase has been studied by measuring the effects of CrADP on the longitudinal nuclear relaxation rate (1/T1) of the protons of water and the protons and phosphorus atom of enzyme-bound glucose-6-P. The paramagnetic effect of CrADP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations measuring this paramagnetic effect at several enzyme concentrations in the presence of glucose-6-P yielded a characteristic enhancement factor for 1/T1 of water protons and the dissociation constant of CrADP from the ternary enzyme . ADPCr . glucose-6-P complex. The latter value (2 mM) is similar to that obtained from kinetic inhibition studies (Danenberg and Cleland [1975]. Biochemistry. 14:28). The presence of glucose-6-P increased the enhancement of the water relaxation rate by enzyme-bound CrADP, suggesting the formation of an enzyme . CrADP . glucose-6-P complex. The existence of such a complex was confirmed by the observation of a paramagnetic effect of enzyme-bound CrADP on the l/T1 of the 31P-nucleus and protons of enzyme-bound glucose-6-P. From the paramagnetic effects of enzyme-bound CrADP on the relaxation rates of the 31P-nucleus and the carbon-bound protons of glucose-6-P in the enzyme . ADPCr . glucose-6-P complex, using the correlation time of approximately 0.7 ns, determined from the magnetic field-dependence of 1/T1 of water protons over the range 24.3-360 MHz, a Cr3+ to phosphorus distance of 6.6 +/- 0.7 A and Cr3+ to alpha- and beta-anomeric proton distances of 8.9 and 9.7 A were calculated. These results imply the absence of a direct coordination of the phosphoryl group of glucose-6-P by the nucleotide-bound metal on hexokinase but indicate van der Waals contact between a phosphoryl oxygen of glucose-6-P and the hydration sphere of the nucleotide-bound metal. The distances are consistent with a model that assumes molecular contact between the phosphorus of glucose-6-P and a beta-phosphoryl oxygen of ADP suggesting an associative phosphoryl transfer. Because after phosphorylation of ADP, the metal ion is coordinated to the transferred phosphoryl group, the overall migration of the phosphoryl group during the phosphoryl transfer is approximately 3.6 A toward the nucleotide-bound metal. Little or no catalysis of phosphoryl transfer from glucose-6-P to alpha, beta-bidentate or beta-monodentate CrADP ( less than or equal to 0.05% of the rate found with MgADP) occurred in the presence of hexokinase, as monitored by glucose formation in a coupled assay system using glucose oxidase and peroxidase. The ability of beta, gamma-bidentate CrATP to act as a substrate (Danenberg and Cleland [1975].
通过测量CrADP对水的质子以及与酶结合的葡萄糖 - 6 - P的质子和磷原子的纵向核弛豫率（1/T1）的影响，研究了Mg - ADP的交换惰性顺磁性类似物CrADP与酵母己糖激酶的相互作用。与酶络合后，CrADP对水质子1/T1的顺磁效应增强。在葡萄糖 - 6 - P存在下，在几种酶浓度下测量这种顺磁效应的滴定产生了水质子1/T1的特征增强因子以及CrADP从三元酶.ADPCr.葡萄糖 - 6 - P复合物的解离常数。后一个值（2 mM）与动力学抑制研究（Danenberg和Cleland [1975]。生物化学。14:28）获得的值相似。葡萄糖 - 6 - P的存在增加了与酶结合的CrADP对水弛豫率的增强，表明形成了酶.CrADP.葡萄糖 - 6 - P复合物。通过观察与酶结合的CrADP对与酶结合的葡萄糖 - 6 - P的31P核和质子的1/T1的顺磁效应，证实了这种复合物的存在。根据与酶结合的CrADP对酶.ADPCr.葡萄糖 - 6 - P复合物中31P核和葡萄糖 - 6 - P的碳结合质子的弛豫率的顺磁效应，使用从24.3 - 360 MHz范围内水质子1/T1的磁场依赖性确定的约0.7 ns的相关时间，计算出Cr3 +与磷的距离为6.6 +/- 0.7 Å，Cr3 +与α - 和β - 异头质子的距离为8.9和9.7 Å。这些结果意味着己糖激酶上核苷酸结合的金属没有直接配位葡萄糖 - 6 - P的磷酰基，但表明葡萄糖 - 6 - P的磷酰氧与核苷酸结合的金属的水合球之间存在范德华接触。这些距离与一个模型一致，该模型假设葡萄糖 - 6 - P的磷与ADP的β - 磷酰氧之间存在分子接触，表明存在缔合性磷酰转移。因为在ADP磷酸化后，金属离子与转移的磷酰基配位，所以在磷酰转移过程中磷酰基向核苷酸结合的金属的总体迁移约为3.6 Å。如在使用葡萄糖氧化酶和过氧化物酶的偶联测定系统中通过葡萄糖形成监测的那样，在己糖激酶存在下，很少或没有发生从葡萄糖 - 6 - P到α，β - 双齿或β - 单齿CrADP的磷酰转移催化（小于或等于用MgADP发现的速率的0.05％）。β，γ - 双齿CrATP作为底物的能力（Danenberg和Cleland [1975]。
