EFFECT OF E20D SUBSTITUTION IN THE ACTIVE-SITE OF ESCHERICHIA-COLI INORGANIC PYROPHOSPHATASE ON ITS QUATERNARY STRUCTURE AND CATALYTIC PROPERTIES

Glutamic acid 20 is an evolutionarily conserved residue found within t he active site of the inorganic pyrophosphatase of Escherichia coli (E -PPase). Here we determine the effect of E20D substitution on the quat ernary structure and catalytic properties of E-PPase. In contrast to w ild-type enzyme, which is hexameric under a variety of conditions, E20 D-PPase can be dissociated by dilution into nearly inactive trimers, a s shown by electrophoresis of cross-linked enzyme, analytical ultracen trifugation, and measurement of catalytic activity as a function of en zyme concentration, Hexamer stability is increased in the presence of both substrate and Mg2+, is maximal at pH 6.5, and falls off sharply a s the pH is lowered or raised from this value. Measured at saturating substrate, 20 mM Mg2+ and pH 7.2, E20D substitution (a) decreases acti vity toward inorganic pyrophosphate (PPi) hydrolysis and oxygen exchan ge between water and inorganic phosphate (P-i), (b) increases the rate of net PPi synthesis, and (c) decreases the amount of enzyme-bound PP i in equilibrium with P-i in solution. Measurements of PPi hydrolysis rate as a function of both Mg2+ concentration and pH for the E20D vari ant show that its decreased activity is largely accounted for on the b asis of an increased pK(a) of the catalytically essential base at the active site, and the need for a Mg2+ stoichiometry of 5 in the enzyme- substrate complex, similar to what is seen for the D97E variant. By co ntrast, wild-type PPase catalysis over a wide range of Mg2+ concentrat ion and pH is dominated by an enzyme-substrate complex having a total of four Mg2+ ions. These results are consistent with a supporting role for Glu20 in PPase catalysis and demonstrate that even conservative m utation at the active site can perturb the quaternary structure of the enzyme.