IDENTIFICATION OF TRANSIENT INTERMEDIATES IN THE BISPHOSPHATASE REACTION OF RAT-LIVER 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE-2,6-BISPHOSPHATASE BY P-31-NMR SPECTROSCOPY

P-31-NMR spectroscopy was used to identify reaction intermediates during catalytic turn-over of the fructose-2,6-bisphosphatase domain (Fru-2,6-P(2)ase) of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. When fructose-2,6-bisphosphate (Fru-2,6-P-2) was added to the enzyme, the P-31-NMR spectrum showed three resonances in addition to those of free substrate: the phosphohistidine (His-P) intermediate, the C-6 phosphoryl group of fructose-6-phosphate bound to the phosphoenzyme, and phosphate generated by the hydrolysis of substrate. Direct analysis of the alkali-denatured phosphoenzyme intermediate by H-1-(31)Pheteronuclear multiple quantum-filtered coherence spectroscopy confirmed the formation of 3-N-phosphohistidine. Binding of fructose 6-phosphate to the bisphosphatase was detected by a down-field shift and broadening of the C-6 phosphoryl resonance. The down-field shift was greater in the presence of the phosphoenzyme intermediate. Inhibition of Fru-2,6-P-2 hydrolysis by fructose 6-phosphate and Fru-2,6-P-2 was shown to involve binding of the sugar phosphates to the phosphoenzyme. This study provides new experimental evidence in support of the reaction mechanism of Fru-2,6-P(2)ase and suggests that the steady-state His-P intermediate exists primarily in the E-P fructose 6-phosphate complex. These results lay a solid foundation for the use of P-31-NMR magnetization transfer studies to provide an in-depth analysis of the bisphosphatase reaction mechanism.