Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase.
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.
Berrisford
John M
JM
The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Hounslow
Andrea M
AM
Akerboom
Jasper
J
Hagen
Wilfred R
WR
Brouns
Stan J J
SJ
van der Oost
John
J
Murray
Iain A
IA
Michael Blackburn
G
G
Waltho
Jonathan P
JP
Rice
David W
DW
Baker
Patrick J
PJ
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005956:Glucose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D046911:Macromolecular Substances; D008670:Metals / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism
C456025:5-phosphoarabinonohydroxamic acid; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D046911:Macromolecular Substances; D008670:Metals; D013403:Sugar Phosphates; D006859:Hydrogen; D005956:Glucose-6-Phosphate Isomerase