Effect of D97E Substitution on the Kinetic and Thermodynamic Properties of Escherichia coli Inorganic Pyrophosphatase

Abstract

Aspartic acid 97 in the inorganic pyrophosphatase of Escherichia coli (E-PPase) has been identified as an evolutionarily conserved residue forming 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.Pi equilibration at pH 7.2 and 8.0, Mg2+ affinity in the presence and absence of substrate, and the Mg2+ and pH dependence of kcat and Km. We find the major effects of D97E substitution are to (a) decrease markedly the pH-independence rates of both PPi hydrolysis and, especially, PPi resynthesis on the enzyme, (b) selectively destabilize both the EMg4PPi complex and the transition state between this complex and the EMg2(MgPi)2 complex, (c) raise the pKa of the basic group "essential" for PPi hydrolysis and for productive PPi binding by 1.5 and > 2.2 log units, respectively, (d) distort a site to which Mg2+ binds in the absence of substrate such that occupancy of the site by Mg2+ no longer confers enzymatic activity, and (e) decrease the affinity of one of the two Mg2+ ions that binds to enzyme in the presence of substrate. That this multiplicity of effects arises from a single Asp to Glu substitution suggests, in the absence of any evidence for a generalized structural change, a tightly integrated active site in which the perturbation induced by conservative substitution at a single location can have widespread functional effects.

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

“…Similar effects of active site substitutions were observed previously in soluble PPases. Whereas catalysis by wild-type E. coli and S. cerevisiae PPases proceeds through both three-and four-metal pathways (28,29), active site variants require the whole complement of four metal ions, but are inactive when only three metal ions bind (30,31). The similarities between the effects of the substitutions in two non-homologous PPase families imply that conserved Glu residues of R-PPase also form part of an active site.…”

Mg2+ activation of Escherichia coli inorganic pyrophosphatase

“…PPase requires four Mg 2+ ions per active site for activity. Two of them are bound directly to the enzyme and the remaining two are bound in the presence of the substrate PPi [1,2]. The active site cavity contains fifteen polar groups, which presumably provide ligands for PPi and Mg 2+ and fulfill other catalytic roles [3,4].…”

“…However, the information on the role of Mg 2+ in catalysis and the mode of its binding to protein is quite limited because the three-dimensional structure has been determined only for the apo-enzyme [3,4]. Estimates of the binding stoichiometry and affinities for Mg 2+ (Kd of 0.08 and 1.7 raM) were obtained using equilibrium dialysis in combination with atomic absorption spectroscopy at pH 7.2 [2], one unit below the pH optimum of PPase activity.…”

Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis?

“…A minimal kinetic scheme, fully accounting for the overall catalysis by E. coli PPase of PPi*Pi equilibration, has been put forward (Baykov et al, 1990;Kapyla et al, 1995). We seek to understand the basis for this catalysis, which accelerates the rate of PPi hydrolysis by 1O' O compared to that in solution (Cooperman, 1982).…”