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. beta-Phosphoglucomutase catalyses the isomerization of beta-glucose-1-phosphate to beta-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 beta-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.
Baxter
Nicola J
NJ
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Olguin
Luis F
LF
Golicnik
Marko
M
Feng
Guoqiang
G
Hounslow
Andrea M
AM
Bermel
Wolfgang
W
Blackburn
G Michael
GM
Hollfelder
Florian
F
Waltho
Jonathan P
JP
Williams
Nicholas H
NH
D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004795:Enzyme Stability; D005459:Fluorides / Q000737:chemistry / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000737:chemistry; D005958:Glucosephosphates / Q000737:chemistry; D013294:Lactococcus lactis / Q000201:enzymology*; D017616:Magnesium Compounds / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010733:Phosphoglucomutase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000577:Amides; D005958:Glucosephosphates; D017616:Magnesium Compounds; D019298:Glucose-6-Phosphate; C031288:magnesium fluoride; C031590:glucose-1-phosphate; D010733:Phosphoglucomutase; D005459:Fluorides