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An inorganic pyrophosphatase is an enzyme that catalyses hydrolysis of free inorganic pyrophosphate into two orthophosphate units. One explicitly balanced choice of ionic forms isActual protonation and metal binding depend on pH and solution conditions. Hydrolysis removes pyrophosphate generated by other reactions, helping couple those reactions to product removal.

In 1952, M. Kunitz isolated crystalline inorganic pyrophosphatase from baker's yeast. The preparation hydrolysed free pyrophosphate, required activating metal ions such as magnesium, and did not interchangeably hydrolyse the tested organic diphosphate-containing compounds. Purifying a catalyst and comparing substrates connected the reaction to a specific protein activity.^[ref]

For a stoichiometric assay example, complete hydrolysis of $10\mu \mathrm{M}$ pyrophosphate adds $20\mu \mathrm{M}$ orthophosphate, provided no other reaction consumes or generates it. Pre-existing phosphate must be measured as background. Stoichiometry predicts product amount; enzyme kinetics, pH and metal availability determine the time required. The yeast preparation's preferred conditions should not be assigned to every inorganic pyrophosphatase.

M. Kunitz isolated crystalline inorganic pyrophosphatase from yeast and characterized its catalytic activity in a 1952 study. The purified protein accelerated hydrolysis of pyrophosphate to separate phosphate units. By testing a purified preparation, the experiment could connect a reaction to a particular enzyme rather than merely observing that an unfractionated biological extract changed its reaction substrate.^[ref]

A revealing result was the requirement for activating metal ions, including magnesium. The reaction rate also depended on pH, temperature and the amounts of enzyme and reaction substrate. Related organic phosphate compounds were not equivalent substrates for this preparation. Thus a shared chemical linkage does not guarantee recognition by the same enzyme. As a simple assay illustration, complete cleavage of 10 micromolar pyrophosphate produces 20 micromolar additional phosphate units. Measuring that increase requires subtracting any phosphate already present; measuring an initial reaction rate further requires a time interval before substantial substrate depletion.^[ref]

Orthophosphate denotes the uncondensed, single-phosphorus phosphate unit. Its fully deprotonated anion is $\mathrm{P}{\mathrm{O}}_4^{3-}$: one phosphorus surrounded by four oxygen atoms. In aqueous biochemical usage, inorganic phosphate, ${\mathrm{P}}_{\mathrm{i}}$, can include protonated forms such as hydrogenphosphate, $\mathrm{HP}{\mathrm{O}}_4^{2-}$, and dihydrogenphosphate, ${\mathrm{H}}_2\mathrm{P}{\mathrm{O}}_4^{-}$. Their proportions depend on pH; the name alone does not specify a unique protonation state.^[ref]

The contrast with pyrophosphate is connectivity. Pyrophosphate joins two phosphorus centres through an oxygen bridge; hydrolysis breaks that bridge to yield two separate orthophosphate units. One charge-balanced representation isThe exact proton balance changes with the chosen chemical species, but two phosphorus atoms remain two phosphorus atoms. M. Kunitz's 1952 isolation of inorganic pyrophosphatase from Saccharomyces cerevisiae provided a purified enzyme that specifically catalysed conversion of inorganic pyrophosphate to orthophosphate, making this distinction experimentally measurable.^[ref]

Pyrophosphate is the two-phosphorus member of the condensed phosphate family, with a phosphorus–oxygen–phosphorus bridge joining its two phosphate units. The fully deprotonated anion is ${\mathrm{P}}_2{\mathrm{O}}_7^{4-}$; its acid is ${\mathrm{H}}_4{\mathrm{P}}_2{\mathrm{O}}_7$. The name also occurs in salts and partially protonated forms, so an actual chemical solution must be described with its pH and protonation state.

Cleaving the bridge by hydrolysis produces two separate phosphate units. One balanced representation isBoth the atoms and the total charge balance: the two products together carry charge $-4$. The equation distinguishes chemical cleavage from merely releasing an intact pyrophosphate ion from a larger molecule.

A classic experiment isolated crystalline inorganic pyrophosphatase from yeast and tested its activity on pyrophosphate. The enzyme accelerated hydrolysis and required activating metal ions, including magnesium. Its failure to hydrolyze the tested organic compounds containing related phosphate linkages demonstrated reaction substrate specificity: recognizing free pyrophosphate is not equivalent to attacking every phosphorus–oxygen–phosphorus linkage.^[ref]