Functional size analysis of pyrophosphatase from <i>Rhodospirillum rubrum</i> determined by radiation inactivation

Wu, Jia Jiu; Tyan, Jing; Pan, Rong

doi:10.1016/0014-5793(91)80552-e

Cited by 17 publications

(12 citation statements)

References 19 publications

(2 reference statements)

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“…Moreover, H ϩ -PPases have recently been identified in the plasma membranes of protozoa (6,7). Both hydrolytic and proton translocation activities are associated with a single polypeptide of 66 -90 kDa (8 -11), which possibly forms a dimer (10,12). H ϩ -PPase is a highly hydrophobic protein, as evident from the 14 -16 transmembrane spans predicted by computer modeling (Fig.…”

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confidence: 95%

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Malinen

Belogurov

Salminen

et al. 2004

Journal of Biological Chemistry

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H ؉ -pyrophosphatase (H ؉ -PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H ؉ -PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu 197 , Glu 202 , Glu 550 , and Glu 649 in the H ؉ -PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP i hydrolysis and PP i -energized proton translocation, indicating that the negative charge of Glu 202 is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg 2؉ concentrations but were only slightly affected at high Mg 2؉ concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two-and three-metal pathways. Substitution of Glu 197 , Glu 202 , and Glu 649 resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu 550 and Glu 649 , R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP i hydrolysis to proton transport.

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confidence: 95%

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Malinen

Belogurov

Salminen

et al. 2004

Journal of Biological Chemistry

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“…The isolated H+-PPase appears to consist of a single polypeptide of 56 kDa (NyrCn et al, 1991), 2-3-times larger than in soluble PPases (Cooperman et al, 1992). The functional unit of H'-PPase in membrane appears to be dimer or trimer (Wu et al, 1991). The activities expressed by this enzyme include, in addition to PP, hydrolysis and synthesis, P,/PP, phosphorous exchange (de Meis et al, 1986) and P,/HOH oxygen exchange (Harvey and Keister, 1981), resulting from partial reversals of the overall reaction.…”

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confidence: 99%

Kinetic Characterization of the Hydrolytic Activity of the H⁺‐Pyrophosphatase of Rhodospirillum rubrum in Membrane‐Bound and Isolated States

Baykov¹,

Sergina²,

Evtushenko³

et al. 1996

European Journal of Biochemistry

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Substrate hydrolysis by the H+-pyrophosphatase (pyrophosphate phosphohydrolase, H+-PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two-pathway reaction scheme in which preformed 1 : 1 and 1 : 2 enzyme . Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+-PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26-32, while having only small effect on substrate binding to EMg,. Formation of EMg requires a basic group with pK, of 7.2-7.7 and confers resistance against inactivation by mersalyl and N-ethylmaleimide to H+-PPase. The dissociation constants governing EMg and EMg, formation, as estimated from the mersalyl-protection assays and steady-state kinetics of PP, hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+-PPase and soluble PPases and the added ability of H+-PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PP, hydrolysis.

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“…H ϩ -PPases represent a distinct class of ion translocases with no sequence similarity to ubiquitous ATP-energized pumps such as F-, V-, and P-type ATPases or ABC transporters (4). Both PPase and proton translocation activities are associated with a single 66 -90-kDa polypeptide (5-7), which possibly forms a dimer (8,9). H ϩ -PPase is a highly hydrophobic protein as estimated from the 14 -16 transmembrane spans predicted by computer modeling (10,11).…”

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confidence: 99%

A Lysine Substitute for K+

Belogurov¹,

Lahti²

2002

Journal of Biological Chemistry

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The H+proton-translocating inorganic pyrophosphatase (H+-PPase) family is composed of two phylogenetically distinct types of enzymes: K+-dependent and K+-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H+-PPase from the thermophilic bacteriumCarboxydothermus hydrogenoformans. Both PPi-hydrolyzing and PPi-energized H+translocation activities of the recombinant enzyme inEscherichia coliinner membrane vesicles are strictly K+-dependent. Here we deduce the K+requirement of all available H+-PPase sequences based on the K+dependence ofC. hydrogenoformansH+-PPase in conjunction with phylogenetic analyses. Our data reveal that K+-independent H+-PPases possess conserved Lys and Thr that are absent in K+-dependent H+-PPases. We further demonstrate that a A460K substitution inC. hydrogenoformansH+-PPase is sufficient to confer K+independence to both PPihydrolysis and PPi-energized H+translocation. In contrast, a A463T mutation does not affect the K+dependence of H+-PPase.

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Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation

Cited by 17 publications

References 19 publications

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Kinetic Characterization of the Hydrolytic Activity of the H⁺‐Pyrophosphatase of Rhodospirillum rubrum in Membrane‐Bound and Isolated States

A Lysine Substitute for K+

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Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation

Cited by 17 publications

References 19 publications

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Kinetic Characterization of the Hydrolytic Activity of the H+‐Pyrophosphatase of Rhodospirillum rubrum in Membrane‐Bound and Isolated States

A Lysine Substitute for K+

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Kinetic Characterization of the Hydrolytic Activity of the H⁺‐Pyrophosphatase of Rhodospirillum rubrum in Membrane‐Bound and Isolated States