The structure of E.coli soluble inorganic pyrophosphatase at 2.7 Å resolution

Abstract

The structure of E.coli soluble inorganic pyrophosphatase has been refined at 2.7 A resolution to an R-factor of 20.9%. The overall fold of the molecule is essentially the same as yeast pyrophosphatase, except that yeast pyrophosphatase is longer at both the N- and C-termini. Escherichia coli pyrophosphatase is a mixed alpha + beta protein with a complicated topology. The active site cavity, which is also very similar to the yeast enzyme, is formed by seven beta-strands and an alpha-helix and has a rather asymmetric distribution of charged residues. Our structure-based alignment extends and improves upon earlier sequence alignment studies; it shows that probably no more than 14, not 15-17 charged and polar residues are part of the conserved enzyme mechanism of pyrophosphatases. Six of these conserved residues, at the bottom of the active site cavity, form a tight group centred on Asp70 and probably bind the two essential Mg2+ ions. The others, more spreadout and more positively charged, presumably bind substrate. Escherichia coli pyrophosphatase has an extra aspartate residue in the active site cavity, which may explain why the two enzymes bind divalent cation differently. Based on the structure, we have identified a sequence motif that seems to occur only in soluble inorganic pyrophosphatases.

Effect of D42N substitution in Escherichia coli inorganic pyrophosphatase on catalytic activity and Mg2+ binding

“…Comparison of these amino at:id sequences reveals little similarity except for 21 residues which are conserved in all known PPases. 15 of them are l¢cated in the PPase active site cavity as shown by X-ray ci '¢stallographic studies of E. coli PPase [4,5], T. thermophilus P~'ase [3] and S. cerevisiae PPase-Mn2+-Pi complex [6]. These d:ta are in accordance with the similarity of the catalytic m~chanisms examined in detail on the E. coli and S. cerevisiae el~zymes [7,8].…”

Dissociation of Hexameric Escherichia coli Inorganic Pyrophosphatase into Trimers on His-136 → Gln or His-140 → Gln Substitution and Its Effect on Enzyme Catalytic Properties

“…coli PPase is homohexameric (Wong et al, 1970) and contains 175 amino acid residues per subunit (Lahti et al, 1988). Its three-dimensional structure, recently been determined at 2.5-2.7-Å resolution (Kankare et al, 1994;Oganessyan et al, 1994), is very like that of S. cerevisiae PPase (Kuranova et al, 1983;Terzyan et al, 1984), in accord with the conservation of active site residues and mechanism in the two enzymes (Cooperman et al, 1992;Kankare et al, 1994). E. coli PPase requires four Mg 2ϩ ions per active site for catalysis, as described in Scheme I.…”

“…7; Kankare et al (1994)). The main components of the interface around the local 2-fold axis are the two antiparallel symmetry-related ␣-helices A (Fig.…”

“…The absence of His residues from the active site cavity (Kankare et al, 1994) and the lack of conserved His residues in soluble PPases (Cooperman et al, 1992) make it clear that His residues have no direct role in catalysis, leaving open the possibility that the chemical modification results arise from an indirect effect. In this work we demonstrate that substitution of each of the five His residues in E. coli PPase with Gln results in substantial retention or even enhancement of catalytic activity, confirming their nonessential character.…”

Solution Structure of the MutT Enzyme, a Nucleoside Triphosphate Pyrophosphohydrolase

“…The a + p fold of MutT resembles, but is not identical to, certain other members of this class of proteins such as the actin binding domain of severin (Schnuchel et al, 1995) and the carbohydrate recognition domain of a mannose binding protein (Weis et al, 1991), both of which bind Ca2+, the phosphotyrosine recognition SH2 domain (Waksman et al, 1992), and the mechanistically related enzyme inorganic pyrophosphatase (Kankare et al, 1994;Teplyakov et al, 1994) although none of these proteins show significant sequence homology to MutT (Mejean et al, 1994).…”