Jarori G K, Mehta A, Kasturi S R, Kenkare U W
Eur J Biochem. 1984 Sep 17;143(3):669-76. doi: 10.1111/j.1432-1033.1984.tb08420.x.
Our previous studies have shown that one manganous ion binds tightly to bovine hexokinase, with a Kd = 25 +/- 4 microM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (epsilon b) is 3.5 at 9 MHz and 23 degrees C [Jarori, G.K. Kasturi, S.R., and Kenkare, U.W. (1981) Arch. Biochem. Biophys. 211, 258-268]. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E X Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme X nucleotide X Mn. Addition of glucose 6-phosphate to enzyme X ATP X Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E X ADP X Mn X Glc-6-P appears to be formed when both the products are present. Beta, gamma-Bidentate Cr(III)ATP has been used to elucidate the role of direct binding of Mn(II) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(III)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron-spin resonance studies on the binding of Mn(II) to the enzyme in the presence of Cr(III)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis. Experiments carried out with hexokinase spin-labeled with 3-(2-iodo-acetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site.
我们之前的研究表明，一个锰离子与牛己糖激酶紧密结合，解离常数Kd = 25±4微摩尔。在9兆赫和23摄氏度下，这种二元复合物（εb）的特征质子弛豫率（PRR）增强为3.5 [贾罗里，G.K. 卡斯图里，S.R.，和肯卡雷，U.W.（1981年）《生物化学与生物物理学文献》211，258 - 268]。基于向这种E×Mn二元复合物中添加核苷酸ATP和ADP时观察到的PRR增强模式，我们现在证明形成了一种核苷酸桥连三元复合物，即酶×核苷酸×Mn。向酶×ATP×Mn中添加6 - 磷酸葡萄糖，会导致ATP - Mn从酶上被竞争性取代。然而，当两种产物都存在时，似乎会形成一种四元复合物E×ADP×Mn×Glc - 6 - P。β，γ - 双齿Cr(III)ATP已被用于阐明Mn(II)直接结合在催化中的作用，以及在核苷酸存在下金属离子与酶相互作用的化学计量关系。双齿Cr(III)ATP作为脑己糖激酶的底物，无需额外的二价阳离子。然而，在Cr(III)ATP存在下对Mn(II)与该酶结合的电子自旋共振研究表明，在核苷酸存在时，两个金属离子与己糖激酶相互作用，一个直接与酶结合，另一个通过核苷酸桥相互作用。正是后者参与催化。用3 - (2 - 碘代乙酰氨基)-2,2,5,5 - 四甲基 - 1 - 吡咯烷基氧基自旋标记的己糖激酶进行的实验清楚地表明，酶上的直接结合Mn位点与其ATP - Mn结合位点明显不同。
