Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers

Abstract

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg 2+ -binding affinity for two of the three sites present in E-PPase (3.5-to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20-to 40-fold). By contrast, Mg 2+ -binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg 2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg 2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer±trimer interface. Mg 2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg 2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.Keywords: pyrophosphatase; magnesium; Tris; quaternary structure; structural modeling.Inorganic pyrophosphatase is a highly efficient catalyst of reversible phosphoryl transfer from the simplest phosphoric acid anhydride, pyrophosphate, to water. Like many similar reactions, it depends on the presence of divalent metal ions, but is remarkable in requiring as many as four such ions per active site. In the product complex Saccharomyces cerevisiae-PPase:manganese phosphate, all four metal ions M1-M4 interact directly with the phosphate molecule P2, and two of them (M3 and M4) also interact with phosphate P1 [1,2]. Metal±PP i interactions appear to shield the negative charge on the electrophile, while both metal ions and hydrogen bond donors lower the pK a of the leaving group in PP i hydrolysis. The metals M1 and M2 [1], or only M2 in an alternative model [2], are believed to be also involved in the activation of the nucleophilic water molecule. Functional studies have indicated that three of the four metal ions are absolutely required for catalysis by S. cerevisiae PPase (Y-PPase) and one, M3 or M4, is modulatory [3±6].Monomers of homohexameric Escherichia coli PPase (E-PPase) are smaller than monomers of homodimeric Y-PPase (175 compared to 286 residues); the primary differences are at the N-and C-termini, which do not take part in the active site. Despite the low overall identity of the primary structures of E-PPase and of the core part of Y-PPase [7], they have very similar folds and active sites [8,9]. Nevertheless, E-PPase binds more Mg 2+ , up to 3 mol´mol 21 in the absence of substrate and 5 mol´mol 21 in its presence [6]. Recent identification of a highaffinity Mg 2+ binding site (M in ...

Reciprocal Effects of Substitutions at the Subunit Interfaces in Hexameric Pyrophosphatase of Escherichia coli

“…5 and Tables I and II, has recently been shown to bind to the active site of E-PPase (21). Tris markedly decreases the k d for H136Q-PPase hexamer, thereby stabilizing the hexameric form (21). In contrast, Tris exerted only a minor effect on the trimer-dimer and dimer-monomer equilibria (data not shown), consistent with the severely decreased substrate binding affinity of these oligomeric forms (see below).…”

“…k cat /K m ) is observed at up to 50 mM Mg 2ϩ concentration. In contrast, k cat /K m is reduced 2.5-to 15-fold for the hexamer and trimer in the presence of 20 mM Mg 2ϩ at pH 9.3-9.5 because of competition between the metal ion bound to M2 and the second metal ion of Mg 2 PP i (10,21). This means that the M2 site also exhibits a markedly decreased affinity in the quadruple variant or that substrate binding becomes dependent on the presence of Mg 2ϩ at both M1 and M2 sites over the whole pH range examined.…”

“…Tris buffer, used in the incubation media for Fig. 5 and Tables I and II, has recently been shown to bind to the active site of E-PPase (21). Tris markedly decreases the k d for H136Q-PPase hexamer, thereby stabilizing the hexameric form (21).…”

“…7). The maximum values of k cat /K m for dimer, trimer (10), and hexamer (21) Table II (21). In trimers, the same is true at pH Ն 8.0, but at pH 7.2 Mg 2ϩ binding to both sites is required (10).…”

A Soluble Pyrophosphatase, a Key Enzyme for Polyphosphate Metabolism in Leishmania

“…Some buffers such as Tris modulate E. coli PPase activity (20) We therefore stored the enzyme in MES/NaOH, pH 6.5, 5 mM MgCl 2 , and 250 mM NaCl. The reactions were then performed in reaction buffers containing either Mg 2ϩ or Zn 2ϩ and at different pH levels.…”