Berrisford John M, Hounslow Andrea M, Akerboom Jasper, Hagen Wilfred R, Brouns Stan J J, van der Oost John, Murray Iain A, Michael Blackburn G, Waltho Jonathan P, Rice David W, Baker Patrick J
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
J Mol Biol. 2006 May 19;358(5):1353-66. doi: 10.1016/j.jmb.2006.03.015. Epub 2006 Mar 24.
The enzymatic aldose ketose isomerisation of glucose and fructose sugars involves the transfer of a hydrogen between their C1 and C2 carbon atoms and, in principle, can proceed through either a direct hydride shift or via a cis-enediol intermediate. Pyrococcus furiosus phosphoglucose isomerase (PfPGI), an archaeal metalloenzyme, which catalyses the interconversion of glucose 6-phosphate and fructose 6-phosphate, has been suggested to operate via a hydride shift mechanism. In contrast, the structurally distinct PGIs of eukaryotic or bacterial origin are thought to catalyse isomerisation via a cis-enediol intermediate. We have shown by NMR that hydrogen exchange between substrate and solvent occurs during the reaction catalysed by PfPGI eliminating the possibility of a hydride-shift-based mechanism. In addition, kinetic measurements on this enzyme have shown that 5-phospho-d-arabinonohydroxamate, a stable analogue of the putative cis-enediol intermediate, is the most potent inhibitor of the enzyme yet discovered. Furthermore, determination and analysis of crystal structures of PfPGI with bound zinc and the substrate F6P, and with a number of competitive inhibitors, and EPR analysis of the coordination of the metal ion within PfPGI, have suggested that a cis-enediol intermediate-based mechanism is used by PfPGI with Glu97 acting as the catalytic base responsible for isomerisation.
葡萄糖和果糖的酶促醛糖 - 酮糖异构化涉及它们的C1和C2碳原子之间氢的转移，原则上可以通过直接氢化物转移或经由顺式 - 烯二醇中间体进行。嗜热栖热菌磷酸葡萄糖异构酶（PfPGI）是一种古菌金属酶，催化6 - 磷酸葡萄糖和6 - 磷酸果糖的相互转化，有人提出它通过氢化物转移机制起作用。相比之下，真核生物或细菌来源的结构不同的PGI被认为通过顺式 - 烯二醇中间体催化异构化。我们通过核磁共振表明，在PfPGI催化的反应过程中，底物和溶剂之间发生了氢交换，排除了基于氢化物转移机制的可能性。此外，对这种酶的动力学测量表明，5 - 磷酸 - D - 阿拉伯糖异羟肟酸酯是尚未发现的该酶最有效的抑制剂，它是假定的顺式 - 烯二醇中间体的稳定类似物。此外，对结合锌和底物F6P以及多种竞争性抑制剂的PfPGI晶体结构的测定和分析，以及对PfPGI中金属离子配位的电子顺磁共振分析表明，PfPGI使用基于顺式 - 烯二醇中间体的机制，其中Glu97作为负责异构化的催化碱基。
