Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases.
Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D-tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS)
D002241:Carbohydrates; D011756:Diphosphates / Q000494:pharmacology*; D007887:Fabaceae; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010732:Phosphofructokinase-1 / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D011424:Propionibacterium / Q000201:enzymology*; D013045:Species Specificity; D013053:Spectrophotometry; D013379:Substrate Specificity
D002241:Carbohydrates; D011756:Diphosphates; D010732:Phosphofructokinase-1