Abstract
The hydrolysis of a phosphate ester can proceed through an intermediate of metaphosphate (dissociative mechanism) or through a trigonal bipryamidal transition state (associative mechanism). Model systems in solution support the dissociative pathway, whereas most enzymologists favor an associative mechanism for enzyme-catalyzed reactions. Crystals of fructose-1,6-bisphosphatase grow from an equilibrium mixture of substrates and products at near atomic resolution (1.3 A). At neutral pH, products of the reaction (orthophosphate and fructose 6-phosphate) bind to the active site in a manner consistent with an associative reaction pathway; however, in the presence of inhibitory concentrations of K+ (200 mm), or at pH 9.6, metaphosphate and water (or OH-) are in equilibrium with orthophosphate. Furthermore, one of the magnesium cations in the pH 9.6 complex resides in an alternative position, and suggests the possibility of metal cation migration as the 1-phosphoryl group of the substrate undergoes hydrolysis. To the best of our knowledge, the crystal structures reported here represent the first direct observation of metaphosphate in a condensed phase and may provide the structural basis for fundamental changes in the catalytic mechanism of fructose-1,6-bisphosphatase in response to pH and different metal cation activators.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Crystals of fructose-1,6bisphosphatase grow from an equilibrium mixture of substrates and products at near atomic resolution (1.3 Å)
FBPase catalyzes the hydrolysis of fructose 1,6-bisphosphate (F16P2) to fructose 6-phosphate (F6P) and orthophosphate (Pi)
Summary
Rfreed Resolution of refinement (Å) Mean B (Å2) for protein Mean B (Å2) for ligands. 0.013 0.033 a Unit cell parameters (a, b, c) in Å for the high pH, high Kϩ and control complexes are (53.600, 83.017, 165.378), (52.813, 82.995, 165.710), and (53.286 82.839 165.599), respectively. b Rsym ϭ ⌺j⌺iIij ϪIj͘/⌺i⌺jIij, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities. c Rfactor ϭ ⌺͉͉Fobs ϪFcalc͉/⌺͉Fobs, ͉Fobs Ͼ 0. d Rfree based upon 10% of the data randomly culled from refinement. Rfreed Resolution of refinement (Å) Mean B (Å2) for protein Mean B (Å2) for ligands. 0.013 0.033 a Unit cell parameters (a, b, c) in Å for the high pH, high Kϩ and control complexes are (53.600, 83.017, 165.378), (52.813, 82.995, 165.710), and (53.286 82.839 165.599), respectively. B Rsym ϭ ⌺j⌺iIij ϪIj͘/⌺i⌺jIij, where i runs over multiple observations of the same intensity, and j runs over all crystallographically unique intensities. C Rfactor ϭ ⌺͉͉Fobs ϪFcalc͉/⌺͉Fobs, ͉Fobs Ͼ 0. D Rfree based upon 10% of the data randomly culled from refinement. Onstrate that FBPase can generate and stabilize metaphosphate at its active site. Variations observed in the mechanism of catalysis by FBPase may arise from changes in the rate-limiting step of a dissociative pathway or even a change in pathway, for instance, from associative to dissociative
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