Effect of Structure Variations in the Inter-subunit Contact Zone on the Activity and Allosteric Regulation of Inorganic Pyrophosphatase from Mycobacterium tuberculosis.

Hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) has a number of structural and functional features that distinguish it from homologous enzymes widely occurring in living organisms. In particular, it has unusual zones of inter-subunit contacts and lacks the N-terminal region common for other PPases. In this work, we constructed two mutant forms of the enzyme, Ec-Mt-PPase and R14Q-Mt-PPase. In Ec-Mt-PPase, the missing part of the polypeptide chain was compensated with a fragment of PPase from Escherichia coli (Ec-PPase). In R14Q-Mt-PPase, a point mutation was introduced to the contact interface between the two trimers of the hexamer. Both modifications significantly improved the catalytic activity of the enzyme and abolished its inhibition by the cofactor (Mg2+ ion) excess. Activation of Mt-PPase by low (~10 µM) concentrations of ATP, fructose-1-phosphate, L-malate, and non-hydrolyzable substrate analogue methylene bisphosphonate (PCP) was observed. At concentrations of 100 µM and higher, the first three compounds acted as inhibitors. The activating effect of PCP was absent in both mutant forms, and the inhibitory effect of fructose-1-phosphate was absent in Ec-Mt-PPase. The effects of other modulators varied only quantitatively among the mutants. The obtained data indicate the presence of allosteric sites in Mt-PPase, which are located in the zones of inter-subunit contact or associated with them.

Enzymes Regulated via Cystathionine ß-Synthase Domains.

Cystathionine ß-synthase (CBS) domains discovered 20 years ago can bind different adenosine derivatives (AMP, ADP, ATP, S-adenosylmethionine, NAD, diadenosine polyphosphates) and thus regulate the activities of numerous proteins. Mutations in CBS domains of enzymes and membrane transporters are associated with several hereditary diseases. The regulatory unit is a quartet of CBS domains that belong to one or two polypeptides and usually form a conserved disk-like structure. CBS domains function as "internal inhibitors" in enzymes, and their bound ligands either amplify or attenuate the inhibitory effect. Recent studies have opened a way to understanding the structural basis of enzyme regulation via CBS domains and widened the list of their bound ligands.

Fast kinetics of nucleotide binding to Clostridium perfringens family II pyrophosphatase containing CBS and DRTGG domains.

We earlier described CBS-pyrophosphatase of Moorella thermoacetica (mtCBS-PPase) as a novel phosphohydrolase that acquired a pair of nucleotide-binding CBS domains during evolution, thus endowing the protein with the capacity to be allosterically regulated by adenine nucleotides (Jämsen, J., Tuominen, H., Salminen, A., Belogurov, G. A., Magretova, N. N., Baykov, A. A., and Lahti, R. (2007) Biochem. J., 408, 327-333). We herein describe a more evolved type of CBS-pyrophosphatase from Clostridium perfringens (cpCBS-PPase) that additionally contains a DRTGG domain between the two CBS domains in the regulatory part. cpCBS-PPase retained the ability of mtCBS-PPase to be inhibited by micromolar concentrations of AMP and ADP and activated by ATP and was additionally activated by diadenosine polyphosphates (AP(n)A) with n > 2. Stopped-flow measurements using a fluorescent nucleotide analog, 2'(3')-O-(N-methylanthranoyl)-AMP, revealed that cpCBS-PPase interconverts through two different conformations with transit times on the millisecond scale upon nucleotide binding. The results suggest that the presence of the DRTGG domain affords greater flexibility to the regulatory part, allowing it to more rapidly undergo conformational changes in response to binding.

Evolutionary conservation of the active site of soluble inorganic pyrophosphatase.

Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi). Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism. PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions. Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism.

Crystal structure of Streptococcus mutans pyrophosphatase: a new fold for an old mechanism.

BACKGROUND: Streptococcus mutans pyrophosphatase (Sm-PPase) is a member of a relatively uncommon but widely dispersed sequence family (family II) of inorganic pyrophosphatases. A structure will answer two main questions: is it structurally similar to the family I PPases, and is the mechanism similar? RESULTS: The first family II PPase structure, that of homodimeric Sm-PPase complexed with metal and sulfate ions, has been solved by X-ray crystallography at 2.2 A resolution. The tertiary fold of Sm-PPase consists of a 189 residue alpha/beta N-terminal domain and a 114 residue mixed beta sheet C-terminal domain and bears no resemblance to family I PPase, even though the arrangement of active site ligands and the residues that bind them shows significant similarity. The preference for Mn2+ over Mg2+ in family II PPases is explained by the histidine ligands and bidentate carboxylate coordination. The active site is located at the domain interface. The C-terminal domain is hinged to the N-terminal domain and exists in both closed and open conformations. CONCLUSIONS: The active site similiarities, including a water coordinated to two metal ions, suggest that the family II PPase mechanism is "analogous" (not "homologous") to that of family I PPases. This is a remarkable example of convergent evolution. The large change in C-terminal conformation suggests that domain closure might be the mechanism by which Sm-PPase achieves specificity for pyrophosphate over other polyphosphates.

Isolation and subunit composition of membrane inorganic pyrophosphatase from rat-liver mitochondria.

The activity of inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) extracted from rat-liver mitochondria with sodium cholate is distributed between three enzyme forms (I-III) which originally reside in the matrix (I) or the inner membrane (II and III). The three forms of pyrophosphatase were separated by means of hydroxyapatite chromatography. The Mr values for enzymes I-III, were 60 000, 120 000 and 210 000, respectively. Membrane pyrophosphatase II, which predominates, was purified to homogeneity. Its molecule consists of two subunits with Mr of 28 000 and 35 000 giving different peptide maps upon BrCN cleavage and polyacrylamide gel electrophoresis. The results suggest that pyrophosphatase II may represent a catalytic part of the membrane pyrophosphatase of these mitochondria.

Fluoride inhibition of inorganic pyrophosphatase. II. Isolation and characterization of a covalent intermediate between enzyme and entire substrate molecule.

A presumed pyrophosphoryl-enzyme intermediate of the reaction catalyzed by bakers' yeast inorganic pyrophosphatase pyrophosphate phosphohydrolase, EC 3.6.1.1) has been isolated using fluoride-mediated inactivation of the enzyme during catalysis. The analysis of the F--inactivated pyrophosphatase revealed the presence of one molecule of PPi and one atom of fluoride per active site. The incubation of the inactivated enzyme at 25 degrees C and pH 7.2 resulted in gradual recovery of catalytic activity and concomitant removal of PPi by a first-order reaction with tau1/2 of 1 h. The digestion of the F--treated pyrophosphatase with pepsin yielded phosphorous-containing peptides, which were reduced with NaBH4 and gave homoserine and homoserine lactone after acid hydrolysis. This suggests that the PPi residue is linked to the protein through a bond of an acyl phosphate type involving the beta-COOH function of aspartic acid. Together with the results of the kinetic studies of fluoride inhibition of pyrophosphatase reported in accompanying papers, these findings strongly indicate that the enzyme-substrate compound stabilized by fluoride is a transient of the catalytic reaction.

Fluoride inhibition of inorganic pyrophosphatase. III. Dependence on the nature of substrate and metal ion cofactor.

Studies of fluoride interaction with bakers' yeast inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) in the presence or absence of enzyme-catalyzed reactions have revealed pronounced specificity of this inhibitor. It was found that the inhibition of enzymic hydrolysis of PPi, ADP, ATP and tripolyphosphate in the presence of Zn2+ and Mn2+ at pH 6.5 is not time dependent and by far less extensive as that observed for the Mg2+-stimulated cleavage of PPi (apparent Ki values differ by three orders of magnitude). Addition of Ca2+ to the latter reaction decrease proportionally the activity of the enzyme and the rate constant for the binding of fluoride to it, which indicates that the enzyme-substrate complexes containing both Mg2+ and Ca2+ are inert in the reaction with fluoride. Preincubation of pyrophosphatase with NaF and various metal cations and substrates, in conditions where the enzyme does not act as a catalyst, does not affect its activity compared to controls lacking fluoride. The results are consistent with the proposed mechanism of mutual hindrance of substrate and fluoride release from the active site of pyrophosphatase.

Fluoride inhibition of inorganic pyrophosphatase. IV. Evidence for metal participation in the active center and a four-site model of metal effect on catalysis.

Atomic spectroscopy of native yeast inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) after gel filtration showed that it only binds activating Mg2% in an easily dissociable manner. Formation of a covalent intermediate between the enzyme and an entire substrate molecular in the presence of fluoride, however, dramatically strengthened the binding of two Mg2+ per subunit and eliminated at neutral pH the effect of added metals on protein fluorescence but not on the absorption spectrum, suggesting that different mental binding sites influence the two spectra. This conclusion was confirmed by spectra studied on native enzyme. A third, low-affinity site for Mg2+ was found on the enzyme pH greater than 8. A model of enzyme-substrate-metal interactions was proposed, according to which the fluorescence-controlling site belongs to the active center and substrate can only be bound to it as a 1 : 1 complex with metals.

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.

Isolation, subunit structure and localization of inorganic pyrophosphatase of heart and liver mitochondria.

A procedure has been developed to isolate separately two forms (I and II) of inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from bovine heart mitochondria with specific activities of 250 and 39 IU/mg, respectively. The values of Mr for enzymes I and II are about 60000 and 185000, respectively. Polyacrylamide gel electrophoresis of pyrophosphatase II in the presence of sodium dodecyl sulfate reveals polypeptides of four types with Mr of 28000 (alpha), 30000 (beta), 40000 (gamma) and 60000 (delta). Enzyme I consists of two subunits similar in mass to alpha and beta. When rat heart and liver mitochondria are fractionated with digitonin and Lubrol WX, pyrophosphatase II, but not I, remains bound to inner membrane fragments. The results show that the two forms of the mitochondrial pyrophosphatase, one of which is localized in the inner membrane, differ in subunit structure but have a common catalytic part.

Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases.

The suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,1-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the Km of the enzyme. The order of inhibitory potency (apparent inhibition constants, Kiapp values, [mu]M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) [almost equal to] ethane-1-hydroxy-1,1-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). The specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the Km of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1, 1-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.

Phosphorylation of rat liver inorganic pyrophosphatase by ATP in the absence and in the presence of protein kinase.

Cytoplasmic inorganic pyrophosphatase of rat liver can be phosphorylated by cAMP-dependent protein kinase on a serine residue with a concomitant increase in enzymic activity. Phosphorylation is also observed in the absence of protein kinase, but in this case much higher concentrations of ATP are required and the stability characteristics of the phosphoenzyme resemble those of an acyl phosphate. Kinase-free phosphorylation of the animal inorganic pyrophosphatase, unlike that of microbial pyrophosphatases, does not activate the enzyme. Pyrophosphatase may thus provide a new example of an enzyme whose evolution involves convergence of regulatory phosphorylation mechanisms.

Tightly bound pyrophosphate in Escherichia coli inorganic pyrophosphatase.

Hexameric inorganic pyrophosphatase of Escherichia coli contains about 1 mol/mol of 'structural' pyrophosphate, which survives gel filtration and prolonged incubation with Mg2+, does not exchange with medium phosphate and pyrophosphate but is removed with 0.8 M perchloric acid. The site of pyrophosphate binding seems to be another than the active site. An additional 0.9 mol of enzyme-bound pyrophosphate is formed in the presence of phosphate and Mg2+ but this pyrophosphate is in fast equilibrium with medium phosphate and appears to be bound to the active site.

Studies on inorganic pyrophosphatase using imidodiphosphate as a substrate.

Baker's yeast inorganic pyrophosphatase has been found to catalyze Mg2+-dependent hydrolysis of imidodiphosphate yielding phosphate and amidophosphate. The reaction proceeds linearly in the presteady state. The catalytic constant is maximal at pH 9.0 and equals 0.5 min-1. Kinetic titrations of the enzyme with imidodiphosphate and Mg2+ have provided direct evidence for the involvement of three Mg2+ per active site in the transition state of the pyrophosphatase reaction.

Oxygen exchange reactions catalyzed by vacuolar H(+)-translocating pyrophosphatase. Evidence for reversible formation of enzyme-bound pyrophosphate.

Vacuolar membrane-derived vesicles isolated from Vigna radiata catalyze oxygen exchange between medium phosphate and water. On the basis of the inhibitor sensitivity and cation requirements of the exchange activity, it is almost exclusively attributable to the vacuolar H(+)-pyrophosphatase (V-PPase). The invariance of the partition coefficient and the results of kinetic modeling indicate that exchange proceeds via a single reaction pathway and results from the reversal of enzyme-bound pyrophosphate synthesis. Comparison of the exchange reactions catalyzed by V-PPase and soluble PPases suggests that the two classes of enzyme mediate P(i)-HOH exchange by the same mechanism and that the intrinsic reversibility of the V-PPase is no greater than that of soluble PPases.

Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes.

An open reading frame located in the COTF-TETB intergenic region of Bacillus subtilis was cloned and expressed in Escherichia coli and shown to encode inorganic pyrophosphatase (PPase). The isolated enzyme is Mn2+-activated, like the authentic PPase isolated from B. subtilis. Although 13 functionally important active site residues are conserved in all 31 soluble PPase sequences so far identified, only two of them are conserved in B. subtilis PPase. This suggests that B. subtilis PPase represents a new family of soluble PPases (a Bs family), putative members of which were found in Archaeoglobus fulgidus, Methanococcus jannaschii, Streptococcus mutans and Streptococcus gordonii.

Cold lability of the mutant forms of Escherichia coli inorganic pyrophosphatase.

The variants of Escherichia coli pyrophosphatase carrying the substitutions Glu20-->Asp, His136-->Gln or His140-->Gln are inactivated, in contrast to the wild-type enzyme, at temperatures below 25 degrees C: their activity measured at 25 degrees C decreases with decreasing the temperature of the stock enzyme solution. The inactivation is completely reversible and is explained by cold-induced dissociation of these hexameric enzymes into less active trimers.

Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme.

1,1-Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H(+)-translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 microM. 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 is concluded that the mitochondrial pyrophosphatase and the H(+)-pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme.

Evolutionary aspects of inorganic pyrophosphatase.

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.