Protein Science, Vol 1, Issue 2 254-258, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

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

I. AUZAT and J. R. GAREL
Laboratoire d'Enzymologie du CNRS, 91198 Gif-sur-Yvette, France

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).
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