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.