Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases

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Abstract
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.degree. 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 of D-psicose-6-phosphate, D-tagatose-6-phosphate, and L-sorbase-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). The bacterial enzyme binds compounds with n = 6,7 and 8 more tightly (Ki .simeq. 200 .mu.M) than any of the others tested. The plant pyrophosphate-dependent phosphofructokinase, however, binds all analogues less tightly than the bacterial enzyme with 1,8-octanediol bisphosphate (K1 .simeq. 650 .mu.M) binding tighter than any of the other analogues in the alkanediol bisphosphate series. Thus, although the active sites for the two enzymes are similar, there are distinct differences.