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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Phosphoglucose isomerase: a ketol isomerase with aldol C2-epimerase activity.
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Phosphoglucose isomerase: a ketol isomerase with aldol C2-epimerase activity.

机译：磷酸葡萄糖异构酶：具有醛醇C2-表异构酶活性的酮醇异构酶。

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摘要

With 13C NMR, phosphoglucose isomerase (PGI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) is shown to produce mannose 6-phosphate (M6P) slowly from a much more rapidly catalyzed equilibrium between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The identity of M6P and its formation from G6P plus F6P are confirmed by 1H NMR and by the ability of PGI to convert M6P to F6P plus G6P. The possibility of contaminating phosphomannose isomerase (PMI, D-mannose-6-phosphate ketol-isomerase, EC 5.3.1.8) is ruled out by finding no exchange of the C1 proton of G6P or of M6P, whereas exchange occurs with a mixture of PMI and PGI in 2H2O. The pro-R and pro-S protons of F6P become the anomeric protons of M6P and G6P through the actions of PMI and PGI, respectively. Both isomerases exchange the C2 proton of their substrate with the medium; hence, when PGI and PMI are added together to hexose phosphate solutions in 2H2O, both the substrate and anomeric protons are exchange rapidly with deuterons from the medium. The rates of C2-epimerization of G6P and M6P by PGI are shown to be proportional to enzyme concentration and inhibited by 5-phosphoarabinoate, a competitive inhibitor of the previously demonstrated isomerase and anomerase activities of PGI. These data show that the epimerization is enzymatically catalyzed and suggest the involvement of the same active site for all three activities. A primary kinetic isotope effect of 7.5 (H/2H) on the rate constant kcat of the M6P C2-epimerase activity was determined by using a coupled enzymatic assay. A model of the mechanism of PGI is offered, which relates C2-epimerase activity to the isomerase and anomerase activities by allowing the cis-enediol intermediate to rotate about the C2-C3 bond axis followed by protonation at C2 but not at C1 from the si face.
机译：借助13C NMR，磷酸葡萄糖异构酶（PGI; D-葡萄糖-6-磷酸酮醇异构酶，EC 5.3.1.9）被证明可以从6-磷酸葡萄糖之间更快得多的催化平衡中缓慢产生6-磷酸甘露糖（M6P）（ G6P）和果糖6-磷酸酯（F6P）。通过1 H NMR和PGI将M6P转化为F6P加G6P的能力，证实了M6P的身份及其由G6P加F6P形成的能力。通过未发现G6P或M6P的C1质子没有交换来排除污染磷酸甘露糖异构酶（PMI，D-甘露糖6-磷酸酮醇异构酶，EC 5.3.1.8）的可能性，而交换是通过PMI混合物进行的和2H2O中的PGI。 F6P的pro-R和pro-S质子分别通过PMI和PGI的作用变成M6P和G6P的异头质子。两种异构酶都用培养基交换其底物的C2质子；因此，当将PGI和PMI一起添加到2H2O中的磷酸己糖溶液中时，底物和异头质子都与来自介质的氘核迅速交换。 PGI对G6P和M6P的C2表观异构化速率与酶浓度成正比，并被5-磷酸氨基阿拉伯酸酯（一种先前证明的PGI异构酶和异构酶活性的竞争性抑制剂）抑制。这些数据表明差向异构酶是酶促催化的，并且暗示了所有三个活性都涉及相同的活性位点。通过使用偶联酶测定法确定了7.5（H / 2H）对M6P C2-表异构酶活性的速率常数kcat的主要动力学同位素效应。提供了一种PGI机制的模型，该模型通过允许顺式-烯二醇中间体绕C2-C3键轴旋转，然后在C2处而不是在C1处使质子化，从而使C2-表异构酶活性与异构酶和异构酶活性相关。面对。

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