Identification of transient intermediates in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by 31P-NMR spectroscopy.
31P-NMR spectroscopy was used to identify reaction intermediates during catalytic turn-over of the fructose-2,6-bisphosphatase domain (Fru-2,6-P2ase) of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. When fructose-2,6-bisphosphate (Fru-2,6-P2) was added to the enzyme, the 31P-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 phospho-enzyme intermediate by 1H-31P heteronuclear 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-P2 hydrolysis by fructose 6-phosphate and Fru-2,6-P2 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-P2ase 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 31P-NMR magnetization transfer studies to provide an in-depth analysis of the bisphosphatase reaction mechanism.
Okar
D A
DA
Department of Biochemistry, School of Medicine, University of Minnesota, Minneapolis 55455, USA.
Kakalis
L T
LT
Narula
S S
SS
Armitage
I M
IM
Pilkis
S J
SJ
D000818:Animals; D001665:Binding Sites; D005636:Fructosephosphates / Q000378:metabolism; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D025481:Phosphofructokinase-2; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins
D005636:Fructosephosphates; D011994:Recombinant Proteins; D006639:Histidine; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; C005436:phosphohistidine