Anomeric specificity of rat hepatic 6-phosphofructo-2-kinase: an NMR study.
The anomeric specificity of 6-phosphofructo-2-kinase for D-fructose-6-phosphate was determined by nuclear magnetic spectroscopy. A mutant 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (His258-Ala) was used to minimize degradation of fructose-2,6-bisphosphate by the bisphosphatase activity. The 1H NMR spectrum of the fructose-2,6-bisphosphate formed from the reaction was identical in the spectral region (3.5 to 4.0 ppm) to that reported for D-fructose-2,6-bisphosphate by Voll et al. (7). The integration of this region accounted for the 7 nonexchangeable protons of the furanose form of fructose. The measured coupling constants and the chemical shifts were identical to those of commercially prepared D-fructose-2,6-bisphosphate. The long range (through 4-bond: P-2, O-2, C-2, C-3, and H-3) coupling between P-2 and H-3, 4JH-3, P-2, was found to be 1.06 Hz and provides strong evidence for the beta-anomer. Additionally, failure to find a similar coupling to the H-la peak ruled out the possibility of existence of the alpha-anomer. These results indicate that only beta-D-fructose-2,6-bisphosphate was synthesized via the 6-phosphofructo-2-kinase reaction. It was concluded that 6-phosphofructo-2-kinase has an absolute stereo specificity for the beta-anomer of D-fructose-6-phosphate.
Lee
Y H
YH
Department of Physiology and Biophysics, State University of New York at Stony Brook, New York 11794, USA.
Picardt
F
F
Pilkis
S J
SJ
D000818:Animals; D005635:Fructosediphosphates / Q000096:biosynthesis*; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D005635:Fructosediphosphates; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C027652:fructose 2,6-diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D025481:Phosphofructokinase-2; D010744:Phosphoric Monoester Hydrolases