l,l-Diphosphonate analogs of pyrophosphate, containmg an amino or a hydroxyl group on the bridge carbon atom, are potent mhibitors of the H*-translocating pyrophosphatases of chromatophores prepared from the bacterium R~udospzr~~Zut~l rubnon and vacuolar membraI~e vesicles prepared from the plant yigna vu&&z. The mhibttton constant for aminomethylenediphosphonate, which bmds competitively with respect to substrate, ts below 2 PM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It 1s concluded that the mttochondrial pyrophosphatase and the H+-pyrophosphatases of ehromatophores and vacuolar membranes belong to two different classes of enzyme.

Substrate hydrolysis by the H+-pyrophosphatase (pyrophosphate phosphohydrolase, H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two-pathway reaction scheme in which preformed 1 : 1 and 1 : 2 enzyme . Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+-PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26-32, while having only small effect on substrate binding to EMg,. Formation of EMg requires a basic group with pK, of 7.2-7.7 and confers resistance against inactivation by mersalyl and N-ethylmaleimide to H+-PPase. The dissociation constants governing EMg and EMg, formation, as estimated from the mersalyl-protection assays and steady-state kinetics of PP, hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+-PPase and soluble PPases and the added ability of H+-PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PP, hydrolysis.

Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme. A series of absorbance versus Pi concentration curves in the presence of 0.5 -20 mM free Mgz+ were obtained at pH 7.2 and computer-fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 2 0.4 mM) used in this analysis was measured with a Mg2+-sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of MgZ + . The Pi-inhibition pattern of pyrophosphate hydolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pibinding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.Inorganic phosphatase catalyzes the reversible reaction of PPi hydrolysis and synthesis and is the simplest member of the class of the enzymes which transfer phosphoryl from a phosphoric acid anhydride. The yeast enzyme is a diiner of identical subunits and its structure has been resolved at 0.3 nm [l]. The enzyme has an absolute requirement for a divalent metal activator such as Mg2+, MnZt, Zn2+ or Co2+ [2, 31. Extensive studies of the hydrolytic reaction catalyzed by yeast pyrophosphatase (for reviews see [4 -61) have revealed a key role of the metal ions in catalysis. The rate of PPi hydrolysis was found to depend on the cube of Mg2+ concentration indicating the involvement of three metal ions/PPi molecule [7, 81, and later binding [9, 131 and X-ray Enzyme. Pyrophosphate phosphohydrolase (EC 3.6.1.1).production upon binding [24]. The present study demonstrates that the overall model for the binding of two Pi molecules to pyrophosphatase active site can be deduced from the effects of Pi on the protein absorption spectrum. The model helps to define the role of the fourth metal ion in PPi synthesis. Furthermore, evidence for a regulatory function of a noncatalytic Pi-binding site is presented. EXPERIMENTAL PROCEDURESHighly purified pyrophosphatase, with a specific activity of 650-700 IU/mg, was obtained from baker's yeast using a modified procedure of Braga et al. [25]. The enzyme, which had been stored as ammonium-sulfate-precipitated pads, was dissolved in 0.1 M Tris/HCI buffer, pH 7.2 (25°C) and freed from ammonium sulfate by passing through a Sephadex G-50 column. The eluate, containing 1-2 mg protein/ml, was supplemented with 2 mM MgC12 and incubated for several days at 4°C. Protein dimer concentration was calculated on the basis of E:&% = 93000 M-' cmpl [2, 261.Difference spectra of Pi complexes ...

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