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- Belogurov, Georgiy A, et al.
(författare)
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H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl
- 2002
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 277:25
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Tidskriftsartikel (refereegranskat)abstract
- H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.
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| 2. |
- Belogurov, Georgiy A, et al.
(författare)
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Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity
- 2005
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 44:6, s. 2088-2096
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Tidskriftsartikel (refereegranskat)abstract
- Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na(+) but displayed the highest activity in the presence of millimolar levels of both Na(+) and K(+). Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na(+) binding sites and one K(+) binding site are involved in enzyme activation. The affinity of the site that binds Na(+) first is increased with increasing K(+) concentration. In contrast, only one Na(+) binding site (K(+)-dependent) and one K(+) binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K(+)-independent Na(+) binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PP(i)-synthesizing activity, which also required Na(+) but was inhibited by K(+). These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na(+)-dependent H(+)-PPase homologues and may be an analogue of Na(+),K(+)-ATPase.
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| 3. |
- Zyryanov, AB, et al.
(författare)
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Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:4, s. 1065-1074
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Tidskriftsartikel (refereegranskat)abstract
- Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn2+ rather than Mg2+, the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and 11 PPases. We measured rates of PPi hydrolysis, PPi synthesis, and P-i/water oxygen exchange catalyzed by sgPPase with Mn2+, Mg2+, or Co2+ in the high-affinity metal-binding site and Mg2+ in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P-i). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PPi complex isomerization known to occur in family I PPases, (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PPi hydrolysis and product release, (c) the marked acceleration of sgPPase catalysis in the presence of Mn2+ and Co2+ results from a combined effect of these ions on bound PPi hydrolysis and P-i release, (d) sgPPase exhibits lower affinity for both PPi and P-i, and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K-m, which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PPi concentrations).
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| 4. |
- Baykov, Alexander A., et al.
(författare)
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Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers
- 1999
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Ingår i: European Journal of Biochemistry. - : Wiley-Blackwell Publishing Inc.. - 0014-2956 .- 1432-1033. ; 260:2, s. 308-317
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Tidskriftsartikel (refereegranskat)abstract
- Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg2+-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg2+-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer–trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.
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