EFFECT OF D97E SUBSTITUTION ON THE KINETIC AND THERMODYNAMIC PROPERTIES OF ESCHERICHIA-COLI INORGANIC PYROPHOSPHATASE

Aspartic acid 97 in the inorganic pyrophosphatase of Escherichia coli (E-PPase) has been identified as an evolutionarily conserved residue f orming part of the active site [Cooperman et al. (1992) Trends Biochem . Sci. 17, 262-266]. Here we determine the effect of D97E substitution on several kinetic and thermodynamic properties of E-PPase, including rate and equilibrium constants for enzyme-catalyzed PPi.P-i equilibra tion at pH 7.2 and 8.0, Mg2+ affinity in the presence and absence of s ubstrate, and the Mg2+ and pH dependence of k(cat) and K-m. We find th e major effects of D97E substitution are to (a) decrease markedly the pH-independence rates of both PPi hydrolysis and, especially, PPi resy nthesis on the enzyme, (b) selectively destabilize both the EMg(4)PP(i ) complex and the transition state between this complex and the EMg(2) (MgPi)(2) complex, (c) raise the pK(a) of the basic group ''essential' ' for PPi hydrolysis and for productive PPi binding by 1.5 and >2.2 lo g units, respectively, (d) distort a site to which Mg2+ binds in the a bsence of substrate such that occupancy of the site by Mg2+ no longer confers enzymatic activity, and (e) decrease the affinity of one of th e two Mg2+ ions that binds to enzyme in the presence of substrate. Tha t this multiplicity of effects arises from a single Asp to Glu substit ution suggests, in the absence of any evidence for a generalized struc tural change, a tightly integrated active site in which the perturbati on induced by conservative substitution at a single location can have widespread functional effects.