Catalytic specificity of yeast inorganic pyrophosphatase for magnesium ion as cofactor. An analysis of divalent metal ion and solvent isotope effects on enzyme function

Welsh, Kathleen A.; Jacobyansky, Andrew; Springs, Bleecker; Cooperman, Barry S.

doi:10.1021/bi00278a029

Cited by 58 publications

(51 citation statements)

References 21 publications

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“…Kinetic studies on Y-PPase (Welsh et al, 1983) and E-PPase provided evidence for a common catalytic mechanism of these enzymes. The model of PP, hydrolysis by PPase proposed by Cooperman et al (1992) assumes general base activation of the attacking nucleophilic water and activation of the leaving phosphoryl group through metal-ion complexation and general acid catalysis.…”

Section: Active Center and Catalytic Mechanismmentioning

confidence: 99%

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus

et al. 1994

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The 3-dimensional structure of inorganic pyrophosphatase from Therrnus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed @sheet and 2 a-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded 0-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the 0-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.

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Section: Active Center and Catalytic Mechanismmentioning

confidence: 99%

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus

et al. 1994

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“…Yeast PPase is a homodimer containing 286 amino acid residues/ monomer (1) and requiring three or four divalent metal ions for catalysis, with Mg 2ϩ conferring the highest activity (2)(3)(4)(5). Two divalent metal ions (M1 and M2) per active site have been identified in the "resting" enzyme by x-ray crystallography and four metal ions (M1-M4) and two phosphates (P1 and P2) in the product complex of Y-PPase (6,7).…”

mentioning

confidence: 99%

Probing Essential Water in Yeast Pyrophosphatase by Directed Mutagenesis and Fluoride Inhibition Measurements

Pohjanjoki¹,

Fabrichniy²,

Kasho³

et al. 2001

Journal of Biological Chemistry

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Inorganic pyrophosphatase (EC 3.6.1.1; PPase) 1 catalyzes reversible phosphoryl transfer from pyrophosphate (PP i ) to water, a metabolically important reaction chemically similar to that catalyzed by numerous ATPases and GTPases. Yeast PPase is a homodimer containing 286 amino acid residues/ monomer (1) and requiring three or four divalent metal ions for catalysis, with Mg 2ϩ conferring the highest activity (2-5). Two divalent metal ions (M1 and M2) per active site have been identified in the "resting" enzyme by x-ray crystallography and four metal ions (M1-M4) and two phosphates (P1 and P2) in the product complex of Y-PPase (6, 7).PP i hydrolysis by PPase occurs via direct attack of water without formation of a phosphorylated enzyme intermediate (8). Modeling the transition state of the chemical step from the structure of the enzyme-product complex Y-PPase⅐Mn 2 (MnP i ) 2 has led to two models that differ in the identity of the water nucleophile placed between metal ions M1 and M2 in the model of Heikinheimo et al. (6) or in the vicinity of Tyr 93 in the model of Harutyunyan et al. (7,9). Although the former model requires an additional "relaxation" step, in which a new water molecule displaces P i oxygen from the position between M1 and M2, it has an advantage of providing an efficient mechanism for nucleophile activation through combined action of the two metal ions and an adjacent Asp 117 residue (see Fig. 1). In aqueous solution and even in crystalline state, proteins are surrounded with water shells, making identification of function-related water molecules a difficult task. Use of fluoride, a potent and most specific inhibitor of cytoplasmic pyrophosphatase, provides a convenient approach to detect such water molecules because molecules of HF and H 2 O are isoelectronic and of similar size, as are the anions derived therefrom.

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“…E. coli PPase requires four Mg 2ϩ ions per active site for catalysis, as described in Scheme I. This scheme, which fully accounts for the overall catalysis of PP i :P i equilibration by E. coli PPase, is a slightly modified version of the one proposed by Baykov et al (1990) following the approach developed for S. cerevisiae PPase (Springs et al, 1981;Welsh et al, 1983).…”

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confidence: 99%

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

Baykov

Dudarenkov

Käpylä

et al. 1995

Journal of Biological Chemistry

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Each of the five histidines in Escherichia coli inorganic pyrophosphatase (PPase) was replaced in turn by glutamine. Significant changes in protein structure and activity were observed in the H136Q and H140Q variants only. In contrast to wild-type PPase, which is hexameric, these variants can be dissociated into trimers by dilution, as shown by analytical ultracentrifugation and cross-linking. Mg 2؉ and substrate stabilize the hexameric forms of both variants. The hexameric H136Q-and H140Q-PPases have the same binding affinities for magnesium ion as wild-type, but their hydrolytic activities under optimal conditions are, respectively, 225 and 110% of wild-type PPase, and their synthetic activities, 340 and 140%. The increased activity of hexameric H136Q-PPase results from an increase in the rate constants governing most of the catalytic steps in both directions. Dissociation of the hexameric H136Q and H140Q variants into trimers does not affect the catalytic constants for PP i hydrolysis between pH 6 and 9 but drastically decreases their affinities for Mg 2 PP i and Mg 2؉. These results prove that His-136 and His-140 are key residues in the dimer interface and show that hexamer formation improves the substrate binding characteristics of the active site.

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Catalytic specificity of yeast inorganic pyrophosphatase for magnesium ion as cofactor. An analysis of divalent metal ion and solvent isotope effects on enzyme function

Cited by 58 publications

References 21 publications

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus

Probing Essential Water in Yeast Pyrophosphatase by Directed Mutagenesis and Fluoride Inhibition Measurements

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

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