The effect of pH on the kinetics of beef-liver fructose bisphosphatase.
1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid; D004305:Dose-Response Relationship, Drug; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005636:Fructosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology
D002729:Chloromercuribenzoates; D005636:Fructosephosphates; D000249:Adenosine Monophosphate; D008345:Manganese; D004228:Dithionitrobenzoic Acid; D006597:Fructose-Bisphosphatase; D008274:Magnesium