Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase

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 ...

Yeast (Saccharomyces cerevisiae) pyrophosphatase (YPPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 M, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and phydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys 83 ), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg 2؉ cofactor, P i product, and the PP i analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k cat remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys 83 residue in the dimer.

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We report here that Escherichia coli pyrophosphatase aggregates in the presence of millimolar Cd 2+ . This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd 2+ concentration. Aggregation was enhanced by Mg 2+ , the natural cofactor of pyrophosphatase, and Mn 2+ .Mutations at the intersubunit metal-binding site had no effect, whereas mutation at Glu139, which is part of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme molecules, suppressed aggregation. These findings indicate that aggregation is affected by Cd 2+ binding to the peripheral metal-binding site, probably by strengthening intermolecular Trp149-Trp149¢ stacking interactions.Keywords: aggregation; cadmium; inorganic pyrophosphatase; site-directed mutagenesis.Protein aggregation is a common phenomenon, with important practical implications. A variety of diseases, including the amyloidoses and prion diseases, as well as other protein deposition disorders, involve protein aggregation [1]. In most cases, the proteins that aggregate are totally or partially unfolded, and the aggregation, which occurs via hydrophobic interactions, is almost completely irreversible [2,3]. Examples of proteins aggregating in their native state, other than salting out and isoelectric point precipitation, are less common, with the aggregation of the mutant hemoglobin that causes sickle-cell anemia being the best known example [4]. In addition, Zn 2+ and other divalent cations have been reported to aggregate native dodecameric glutamine synthethase into tubular structures [5] and to have a role in amyloid formation [6,7].Escherichia coli inorganic pyrophosphatase (PPase) is an essential enzyme that converts pyrophosphate, a byproduct of many biosynthetic reactions, into phosphate [8]. The native PPase molecule is formed by six identical subunits, of 20 kDa each, arranged in parallel layers of trimers [9,10] , a common polluting ion that is toxic to E. coli at millimolar concentrations [14], and we describe the mechanism behind this aggregation. Materials and methodsWild-type E. coli PPase and PPase variants were prepared and purified as described previously [15]. The final preparations were homogeneous, according to SDS/PAGE. Enzyme aggregation was followed by measuring the absorbance of enzyme solution at 440 nm in a quartz cuvette of 1 cm path length. In kinetic experiments, an aliquot of 0.1 M cadmium acetate solution was added to 0.7 mL of enzyme solution (0.37 mgAEmL )1 ) containing 0.1 M Tris/HCl (pH 7.2), 1 mM MgCl 2 and 5 mM dithiothreitol; the contents of the cuvette were rapidly mixed and the absorbance was recorded on a Pharmacia-LKB Ultrospec Plus spectrophotometer. In titration experiments, metal salt was added in 1.4 lL increments, the contents of the cuvette were stirred for 3 min, and the absorbance of the solution was measured. This time was selected on the basis of the observation that 3 min was sufficient for aggregation to r...