MgF(3)(-) and alpha-galactose 1-phosphate in the active site of beta-phosphoglucomutase form a transition state analogue of phosphoryl transfer.
(19)F-based NMR analysis and hydrogen/deuterium primary isotope shifts establish the formation of a highly populated solution-state trigonal bipyramidal complex involving beta-phosphoglucomutase (beta-PGM), alpha-galactose 1-phosphate (alphaGal1P), and trifluoromagnesate (MgF(3)(-)), PGM-MgF(3)-alphaGal1P, that is a transition state analogue for phosphoryl transfer. Full backbone resonance assignment of the protein shows that its structure is in the closed conformation required for catalytic activity and is closely related to the corresponding complex with glucose 6-phosphate, which we have recently identified using NMR analysis in solution and X-ray crystallography in the solid state. The previous identification of three structural waters in a PGM-alphaGal1P binary substrate complex had indicated that, in the presence of alphaGal1P, magnesium ions, and fluoride, beta-PGM should indeed form a PGM-MgF(3)-alphaGal1P-TSA complex whereas, in the solid-state, apparently it did not. This cast doubt on the validity of the interpretation of MgF(3)(-) complexes. The present work establishes that, in solution, the expectation that a PGM-MgF(3)-alphaGal1P-TSA complex should readily form is fulfilled. These results thus refute the final evidence used to claim that the trigonal bipyramidal species observed in some solid-state structures of complexes involving beta-PGM are pentaoxyphosphorane intermediates.
Baxter
Nicola J
NJ
Department of Molecular Biology & Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Hounslow
Andrea M
AM
Bowler
Matthew W
MW
Williams
Nicholas H
NH
Blackburn
G Michael
GM
Waltho
Jonathan P
JP
D020134:Catalytic Domain; D005459:Fluorides / Q000737:chemistry* / Q000378:metabolism; D005958:Glucosephosphates / Q000737:chemistry*; D017616:Magnesium Compounds / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D010733:Phosphoglucomutase / Q000737:chemistry* / Q000378:metabolism
D005958:Glucosephosphates; D017616:Magnesium Compounds; D010710:Phosphates; C031590:glucose-1-phosphate; D010733:Phosphoglucomutase; D005459:Fluorides