Use of 3-D computer modelling and kinetic studies to analyse grapefruit pyrophosphate-dependent phosphofructokinase.
The glycolytic reaction of grapefruit PPi-dependent phosphofructokinase (PFP) depends on the presence of Fru-2,6-P2 (Ka = 6.7 nM). This molecule was further demonstrated in grapefruit juice sac cells. Citrate, alpha-ketoglutarate and isocitrate competitively inhibited the binding of Fru-2,6-P2 to PFP. The affinity for Fru-6-P (Km = 159 microM) and PPi (Km = 33 microM) were not affected by the addition of these molecules. In the gluconeogenic reaction, the presence of Fru-2,6-P2 did not affect the Km of Fru-1,6-P2 (61 microM) in contrast to orange fruit PFP. These results led to the building of a computer model of PFP, based on the known structure of Bacillus stearothermophilus ATP-dependent phosphofructokinase (ATP-PFK). The results show that catalysis of Fru-6-P in the alpha chain is most unlikely, due to amino-acid substitutions and that Fru-2,6-P2 can bind between the alpha and beta subunits.
Van Praag
E
E
Kennedy-Leigh Centre for Horticultural Research, Hebrew University of Jerusalem, Rehovot, Israel.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D019343:Citric Acid / Q000378:metabolism / Q000494:pharmacology; D002957:Citrus / Q000201:enzymology*; D005635:Fructosediphosphates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation; D011198:Solanum tuberosum / Q000201:enzymology
D005635:Fructosediphosphates; D007656:Ketoglutaric Acids; D019343:Citric Acid; D000244:Adenosine Diphosphate; C027652:fructose 2,6-diphosphate; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase