Metaphosphate in the active site of fructose-1,6-bisphosphatase.
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.
Choe
Jun-Yong
JY
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Iancu
Cristina V
CV
Fromm
Herbert J
HJ
Honzatko
Richard B
RB
D001665:Binding Sites; D003460:Crystallization; D006597:Fructose-Bisphosphatase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010710:Phosphates / Q000737:chemistry*
D010710:Phosphates; D006597:Fructose-Bisphosphatase