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

doi:10.1073/pnas.0604448103

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

^†Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom;
^‡Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom;
^¶Centre for Chemical Biology, Department of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom; and
^‖Bruker BioSpin GmbH, Silberstreifen 4, 76287 Rheinstetten, Germany

Edited by Perry A. Frey, University of Wisconsin, Madison, WI, and approved July 26, 2006 (received for review May 30, 2006)

Abstract

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 ¹⁹F-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 MgF₃ ⁻ mimics the transferring PO₃ ⁻ 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.

Footnotes

^††To whom correspondence may be addressed. E-mail: fh111{at}cam.ac.uk, j.waltho{at}sheffield.ac.uk, or n.h.williams{at}sheffield.ac.uk
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.
The authors declare no conflict of interest.
This paper was submitted directly (Track II) to the PNAS office.
Data deposition: The NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession nos. 7234 and 7235).
Abbreviations:

TS,

transition state;

TSA,

TS analogue;

β-PGM,

β-phosphoglucomutase;

β-G1P,

β-glucose-1-phosphate;

β-G16BP,

β-glucose-1,6-bisphosphate;

G6P,

glucose-6-phosphate;

INT,

intermediate