Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase‐1

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Abstract
In the presence of its allosteric activator GDP, the major phosphofructokinase‐1 from Escherichia coli K12 follows Michaelis—Menten kinetics. The kinetic behavior observed at steady‐state using different concentrations of the substrates ATP and fructose‐6‐phosphate and the pattern of inhibition by the substrate analogs adenylyl‐(β,γ‐methylene)‐diphosphonate and D‐arabinose‐5‐phosphate are consistent with a random sequential mechanism in rapid equilibrium, rather than with an ordered binding as was suggested earlier. However, ATP and fructose‐6‐phosphate do not bind independently to the same active site, since the apparent affinity for one substrate is decreased about 20‐fold when the other substrate is already bound. The antagonism between ATP and fructose‐6‐phosphate shows that a negative interaction occurs during the reaction with E. coli phosphofructokinase‐1 which must be considered in addition to its allosteric properties.