pH dependence of the reverse reaction catalyzed by phosphofructokinase I from Escherichia coli : Implications for the role of Asp 127

Abstract The kinetics of the reverse reaction catalyzed by Escherichia coli phosphofructokinase, i.e., the synthesis of ATP and fructose‐6‐phosphate from ADP and fructose‐1,6‐bisphosphate, have been studied at different pH values, from pH 6 to pH 9.2. Hyperbolic saturations of the enzyme are observed for both substrates. The affinity for fructose‐1,6‐bisphosphate decreases with pH following the ionization of a group with a pK of 6.6, whereas the catalytic rate constant and perhaps the affinity for ADP are controlled by the ionization of a group with a pK of 6. Several arguments show that the pK of 6.6 is probably that of the carboxyl group of Asp 127, whereas the pK of 6 is tentatively attributed to the carboxyl group of Asp 103. The pK of 6.6 is assigned to the carboxyl group of Asp 127 in the free enzyme, and a simple model suggests that the same group would have an abnormally high pK, above 9.6, in the complex between phosphofructokinase and fructose‐1,6‐bisphosphate. It is proposed that the large pK shift of more than 3 pH units upon binding of fructose‐1,6‐bisphosphate is due to an electrostatic repulsion that could exist between the 1‐phosphate group and the carboxyl group of Asp 127, which are close to each other in the crystal structure of phosphofructokinase (Shirakihara, Y.&Evans, P.R., 1988, J. Mol. Biol. 204 , 973–994). The same interpretation would also explain the much higher affinity of the enzyme for fructose‐1,6‐bisphosphate when Asp 127 is protonated. Because of its pK above 9.6, Asp 127 would be protonated in the complex between phosphofructokinase and fructose‐1,6‐bisphosphate, and could act as a proton donor, in agreement with its catalytic role as a proton acceptor in the forward reaction (Hellinga, H.W.&Evans, P.R., 1987, Nature 327 , 437–439).