Interaction of <i>Escherichia coli</i> inorganic pyrophosphatase active sites

Avaeva, S.M.; Grigorjeva, Olga; Mitkevich, Vladimir A.; Sklyankina, V. A.; Varfolomeyev, S.D.

doi:10.1016/s0014-5793(99)01686-5

Cited by 13 publications

(10 citation statements)

References 13 publications

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“…The A 620 was measured. [25,26] Characterization of UMK: The activity was determined by using a coupled spectrophotometry assay. The reaction was carried out in a reaction solution (1 mL) that contained Tris/HCl (50 mM, pH 7.4), KCl (50 mM), MgCl 2 (2 mM), ATP (4 mM), UMP (2 mM), phosphoenolpyruvate (PEP; 1 mM), NADH (0.2 mM), pyruvate kinase (2 U), and lactate dehydrogenase (2 U).…”

Section: Methodsmentioning

confidence: 99%

Combined Biosynthetic Pathway For De Novo Production of UDP-Galactose: Catalysis with Multiple Enzymes Immobilized on Agarose Beads

Liu

Zhang

Chen³

et al. 2002

ChemBioChem

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Regeneration of sugar nucleotides is a critical step in the biosynthetic pathway for the formation of oligosaccharides. To alleviate the difficulties in the production of sugar nucleotides, we have developed a method to produce uridine diphosphate galactose (UDP-galactose). The combined biosynthetic pathway, which involves seven enzymes, is composed of three parts: i) the main pathway to form UDP-galactose from galactose, with the enzymes galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophosphorylase, and inorganic pyrophosphatase, ii) the uridine triphosphate supply pathway catalyzed by uridine monophosphate (UMP) kinase and nucleotide diphosphate kinase, and iii) the adenosine triphosphate (ATP) regeneration pathway catalyzed by polyphosphate kinase with polyphosphate added as an energy resource. All of the enzymes were expressed individually and immobilized through their hexahistidine tags onto nickel agarose beads ("super beads"). The reaction requires a stoichiometric amount of UMP and galactose, and catalytic amounts of ATP and glucose 1-phosphate, all inexpensive starting materials. After continuous circulation of the reaction mixture through the super-bead column for 48 h, 50 % of the UMP was converted into UDP-galactose. The results show that de novo production of UDP-galactose on the super-bead column is more efficient than in solution because of the stability of the immobilized enzymes.

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Section: Methodsmentioning

confidence: 99%

Combined Biosynthetic Pathway For De Novo Production of UDP-Galactose: Catalysis with Multiple Enzymes Immobilized on Agarose Beads

Liu

Zhang

Chen³

et al. 2002

ChemBioChem

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“…Hydrolysis of MgPP i by this form did not obey the Michaelis-Menten equation, resulting in nonlinear Lineweaver-Burk plots. This finding had been first explained by a negative coop erativity of the two active sites within a trimer differing with their affinities with respect to MgPP i [14]. Analogous behavior was later observed for the trimeric and hexameric forms of the mutant E PPase Asp26Ala with the substitution in the intertrimeric interface.…”

mentioning

confidence: 73%

Metal-free PPi activates hydrolysis of MgPPi by an Escherichia coli inorganic pyrophosphatase

Vainonen

Vorobyeva

Rodina

et al. 2005

Biochemistry (Moscow)

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Soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) is a hexamer forming under acidic conditions the active trimers. We have earlier found that the hydrolysis of a substrate (MgPP(i)) by the trimers as well as a mutant E-PPase Asp26Ala did not obey the Michaelis-Menten equation. To explain this fact, a model has been proposed implying the existence of, aside from an active site, an effector site that can bind PP(i) and thus accelerate MgPP(i) hydrolysis. In this paper, we demonstrate that the noncompetitive activation of MgPP(i) hydrolysis by metal-free PP(i) can also explain kinetic features of hexameric forms of both the native enzyme and the specially obtained mutant E-PPase with a substituted residue Glu145 in a flexible loop 144-149. Aside from PP(i), its non-hydrolyzable analog methylene diphosphonate can also occupy the effector site resulting in the acceleration of the substrate hydrolysis. Our finding that two moles of [32P]PP(i) can bind with each enzyme subunit is direct evidence for the existence of the effector site in the native E-PPase.

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“…It has been reported that there is an equilibrium between trimeric and hexameric forms of PPase. The trimeric form of PPase exhibited different catalytic activity compared to the WT enzyme [41,42]. Gel filtration demonstrated that Na + , Mg 2+ , and PPi do not greatly alter the global fold of AbPPase (Figure 8A,B).…”

Section: Resultsmentioning

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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Interaction of Escherichia coli inorganic pyrophosphatase active sites

Cited by 13 publications

References 13 publications

Combined Biosynthetic Pathway For De Novo Production of UDP-Galactose: Catalysis with Multiple Enzymes Immobilized on Agarose Beads

Combined Biosynthetic Pathway For De Novo Production of UDP-Galactose: Catalysis with Multiple Enzymes Immobilized on Agarose Beads

Metal-free PPi activates hydrolysis of MgPPi by an Escherichia coli inorganic pyrophosphatase

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

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