Inorganic pyrophosphatase

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Introduction

Inorganic pyrophosphatases (PPases) are essential enzymes that play an important role in controlling the cellular concentration of inorganic pyrophosphate (PPi), which drives biosynthetic reactions such as nucleic acid and protein synthesis. PPi is formed in large quantities as a by-product of biosynthetic reactions, and its concentration affects the intracellular homeostasis of these physiological reactions. PPase catalyzes the simplest phosphoryl transfer reaction, the hydrolysis of a symmetric pyrophosphate (PPi) substrate to two inorganic phosphate (Pi) molecules. Of the four distinct classes of PPases (membrane-integral M PPases and soluble family I, family II, and family III PPases), family III S-PPases have not been extensively characterized and are only present in a few bacterial species. The other three PPase families thus represent enzyme solutions primarily or exclusively dedicated to pyrophosphatase catalysis.

Commonalities and differences

Depending on the enzyme family, pH, and PPi concentration, three or four metal ions may be required for catalysis by PPases. The catalysis is based on leaving group activation and effective nucleophile generation, and proceeds in the absence of an enzyme-phosphate intermediate. This suggests that catalysis all occurs within the inorganic metal phosphate cages, with protein side chains mainly playing an auxiliary role. In terms of pyrophosphate hydrolysis, the three biggest differences between the various enzymes are their different responses to two inhibitors: fluoride, and the diphosphonates, especially to aminomethylenediphosphonate (AMDP), and the desired divalent cation type. For catalysis, M-PPases are the slowest (kcat≈10s^-1), while family I PPases are about an order of magnitude higher (kcat≈200s^-1), and family II enzymes are an order of magnitude higher than the activity of family I enzymes (kcat≈2000s^-1).

Soluble PPases, especially family I enzymes, represent a better-understood phosphotransferase due to the availability of detailed structural and functional information. The biological functions of family I and family II PPases are identical, their sole purpose is to remove pyrophosphate. But their sequences and structures are unrelated, and the mechanisms appear to be different. Membrane integral PPases (M-PPases) are very different from their soluble counterparts. M-PPases also have broader biological functions than soluble PPases, as they play a role in the survival of plants and bacteria under various low-energy stress conditions.

Table 1. Selected kinetic properties of PPases and their inhibition by F^- and AMDP

Structure and mechanism of soluble pyrophosphatases

Family I PPases, first crystallized in 1952, have been extensively studied, especially PPases from Escherichia coli (EPPase) and Saccharomyces cerevisiae (YPPase). Family I PPases present in all kingdoms of life, fold into a compact single domain structure with a conserved five-stranded OB-fold β-barrel at its core, with the active site at the top. Eukaryotic enzymes usually exist as dimers, while bacterial enzymes are usually hexamers. Although sequence conservation in family I PPases hardly extends beyond the 20 charged and hydrophilic residues in the active site, they can be reliably identified by the conserved D-(S/G/N)-D-P-ali-D-ali-ali motifs (ali = C/I/L/M/V). The striking features of catalysis of family I PPases are that all lone pairs in the substrate are coordinated, and that PPi hydrolysis proceeds via an associative mechanism in which the electrophilic phosphate moiety of PPi is attacked by nucleophiles of activated water molecules.

Figure 1. Side by side comparison of overall ribbon views of substrate bound conformations of Family I yeast PPase (YPPase, left) (1E6A), Family II B. subtilis PPase (BsPPase, middle) (2HAW) and H⁺-pumping mung bean PPase (VrPPase, right) (Kajander, T.; et al. 2013)

Family II PPases exist in bacterial and archaeal lineages, particularly Bacilli and Clostridia, including several human pathogens. Family II PPases are structurally distinct from family I PPases, and the two-domain proteins of these homodimers belong to the "DHH" phosphatase superfamily, where DHH refers to a conserved Asp-His-His motif in the N-terminal domain that is critical for metal ion binding specificity. The active site of family II PPases is located between the N-terminal DHH and C-terminal DHHA2 domains. As mentioned above, family II PPases show the highest catalytic activity in the presence of metal ions Mn²⁺ or Co²⁺. The binding and kinetic preferences of transition metals can be explained according to the architecture of the active site.

Figure 2. Side by side comparison of the hydrolytic sites of YPPase (left), BsPPase (middle) and VrPPase (right), emphasising the difference in detailed active site geometry between the three enzymes (Kajander, T.; et al. 2013)