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

Baykov, Alexander A.; Bakuleva, Natalia P.; Nazarova, T. I.; Avaeva, S.M.

doi:10.1016/0005-2744(77)90150-4

Cited by 25 publications

(20 citation statements)

References 19 publications

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“…On the other hand, the bound PP i cannot hydrolyze and leave as P i . This explains why fluoride stabilizes the enzyme-substrate intermediate to an extent allowing its isolation by gel filtration (30). At neutral pH, the bridging water molecule exists predominantly as OH Ϫ in EM 4 PP and, presumably, EM 4 P 2 (21), which explains their very slow, if any, binding of the negatively charged fluoride ion.…”

Section: Roles For the Six Residues In Metal Binding And Catalysis-mentioning

confidence: 99%

“…Fluoride Binding Site-The slow inhibition step results in the incorporation of one fluoride ion per substrate molecule, which remains intact and tightly bound to PPase (30), suggesting that fluoride replaces the nucleophilic water. Substitutions mispositioning its ligands are thus expected to affect fluoride binding.…”

Section: Roles For the Six Residues In Metal Binding And Catalysis-mentioning

confidence: 99%

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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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Section: Roles For the Six Residues In Metal Binding And Catalysis-mentioning

confidence: 99%

Section: Roles For the Six Residues In Metal Binding And Catalysis-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

Self Cite

View full text Add to dashboard Cite

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“…Inhibition of yeast PPase by fluoride has been extensively studied. It has been proposed that fluoride strengthens the binding of the enzyme to the Mg-PP i complex, resulting in a covalent bond (Baykov et al 1976(Baykov et al , 1977. However, not all PPases interact with fluoride in the same manner.…”

Section: Discussionmentioning

confidence: 98%

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

Oliva

Romero

Ayala

et al. 2000

Archives of Microbiology

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A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K(m) for Mg-PP(i)2 hydrolysis of 90 microM, and a Vmax estimated at 24.0 micromol P(i) min(-1) mg(-1).

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“…Even though Cohn. M. initially developed a model for the PPase catalytic mechanism that proposed a covalent phosphorylated enzyme intermediate [26], evidence for this intermediate was not reported until the 1970s [28]. Therefore, a new model was proposed [29] in which a water molecule was coordinated and activated by M1 and M2 as the nucleophile [30] that could attack PPi and thus destroy the covalent bond between the two Pis.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, a new model was proposed [29] in which a water molecule was coordinated and activated by M1 and M2 as the nucleophile [30] that could attack PPi and thus destroy the covalent bond between the two Pis. In addition, fluoride, which has similar properties to hydroxide, could inhibit PPase catalytic activity, but not all of it [28]. Co-crystal structures of PPases with fluorides showed that the fluoride atoms could occupy positions of the nucleophilic water [22].…”

Section: Introductionmentioning

confidence: 99%

Crystal Structures of Pyrophosphatase from Acinetobacter baumannii: Snapshots of Pyrophosphate Binding and Identification of a Phosphorylated Enzyme Intermediate

Wang

Yang³

et al. 2019

IJMS

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All living things have pyrophosphatases that hydrolyze pyrophosphate and release energy. This energetically favorable reaction drives many energetically unfavorable reactions. An accepted catalytic model of pyrophosphatase shows that a water molecule activated by two divalent cations (M1 and M2) within the catalytic center can attack pyrophosphate in an SN2 mechanism and thus hydrolyze the molecule. However, our co-crystal structure of Acinetobacter baumannii pyrophosphatase with pyrophosphate shows that a water molecule from the solvent may, in fact, be the actual catalytic water. In the co-crystal structure of the wild-type pyrophosphatase with pyrophosphate, the electron density of the catalytic centers of each monomer are different from one another. This indicates that pyrophosphates in the catalytic center are dynamic. Our mass spectroscopy results have identified a highly conserved lysine residue (Lys30) in the catalytic center that is phosphorylated, indicating that the enzyme could form a phosphoryl enzyme intermediate during hydrolysis. Mutation of Lys30 to Arg abolished the activity of the enzyme. In the structure of the apo wild type enzyme, we observed that a Na+ ion is coordinated by residues within a loop proximal to the catalytic center. Therefore, we mutated three key residues within the loop (K143R, P147G, and K149R) and determined Na+ and K+-induced inhibition on their activities. Compared to the wild type enzyme, P147G is most sensitive to these cations, whereas K143R was inactive and K149R showed no change in activity. These data indicate that monovalent cations could play a role in down-regulating pyrophosphatase activity in vivo. Overall, our results reveal new aspects of pyrophosphatase catalysis and could assist in the design of specific inhibitors of Acinetobacter baumannii growth.

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

Cited by 25 publications

References 19 publications

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

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

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

Crystal Structures of Pyrophosphatase from Acinetobacter baumannii: Snapshots of Pyrophosphate Binding and Identification of a Phosphorylated Enzyme Intermediate

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