A Trojan horse transition state analogue generated by MgF3− formation in an enzyme active site

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of β-glucose-1-phosphate to β-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 103; no. 40; pp. 14732 - 14737
Main Authors Baxter, Nicola J, Olguin, Luis F, Golicnik, Marko, Feng, Guoqiang, Hounslow, Andrea M, Bermel, Wolfgang, Blackburn, G Michael, Hollfelder, Florian, Waltho, Jonathan P, Williams, Nicholas H
Format Journal Article
LanguageEnglish
Published United States National Acad Sciences 03.10.2006
National Academy of Sciences
Subjects
Amides - chemistry
Binding Sites
Biochemistry
Biological Sciences
Catalysis
Enzyme kinetics
Enzyme Stability
Fluorides - chemistry
Fluorides - metabolism
Glucose-6-Phosphate - chemistry
Glucosephosphates - chemistry
Lactococcus lactis - enzymology
Magnesium Compounds - chemistry
Magnesium Compounds - metabolism
NMR
Nuclear magnetic resonance
Nuclear Magnetic Resonance, Biomolecular
Phosphates
Phosphoglucomutase - antagonists & inhibitors
Phosphoglucomutase - chemistry
Phosphoglucomutase - metabolism
Physical Sciences
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Summary:Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of β-glucose-1-phosphate to β-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19 F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF 3 − mimics the transferring PO 3 − moiety. Here we present a detailed characterization of the metal ion–fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of β-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography. enzyme mechanism fluoride inhibition NMR structure phosphoryl transfer isosteric isoelectronic transition state analogue
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Edited by Perry A. Frey, University of Wisconsin, Madison, WI, and approved July 26, 2006
Author contributions: N.J.B., L.F.O., and M.G. contributed equally to this work; N.J.B., L.F.O., M.G., A.M.H., G.M.B., F.H., J.P.W., and N.H.W. designed research; N.J.B., L.F.O., M.G., A.M.H., W.B., J.P.W., and N.H.W. performed research; G.F. and W.B. contributed new reagents/analytic tools; N.J.B., L.F.O., M.G., F.H., J.P.W., and N.H.W. analyzed data; and N.J.B., L.F.O., M.G., G.M.B., F.H., J.P.W., and N.H.W. wrote the paper.
Present address: Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0604448103