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Jeeves, Mark; Kj, Smith; Pg, Quirk; Np, Cotton; J, Baz Jackson

doi:10.1023/a:1008300609352

Cited by 16 publications

(10 citation statements)

References 4 publications

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“…However, experiments indicated that the binding constant is similar, within a factor of 3, in isolated dI (34), and in dI⅐dIII complexes. 3 Because the value is quite high, measurement of K d from the quenching of Trp 72 fluorescence suffers from significant error due to the inner-filtering effect of the added nucleotide (11,30). Nevertheless, results from this kind of experiment strongly supported the conclusion that the K d of dI for NADH is unaffected by complexation with dIII (data not shown).…”

Section: Figmentioning

confidence: 80%

“…The apparent first order rate constants for phase A (558 and 610 s Ϫ1 ) and for phase B (32 and 28 s Ϫ1 ) were similar for the wild-type and the mutant dIII proteins. We previously showed that the steady-state kinetics of transhydrogenation are not significantly affected by this amino acid substitution (20) and, furthermore, 1 H, 15 N HSQC NMR spectra of rrdIII.E155W (in the NADP ϩ and NADPH forms) differed from spectra of wild-type protein (25,30) only in those resonances that are assigned to residues close to the site of the mutation (data not shown).…”

Section: Single-turnover Transhydrogenation Revealed By Changes Inmentioning

confidence: 84%

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Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Venning

Peake²,

Quirk

et al. 2000

Journal of Biological Chemistry

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Transhydrogenase, which is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria, catalyzes the reaction shown below.A single proton is translocated across the membrane, from the p-aqueous phase (the "outside" of intact mitochondria and bacteria) to the n-aqueous phase (the "inside"), for each hydride equivalent transferred from NADH to NADP ϩ . Under most conditions, this is the in vivo direction; the reaction is driven by the proton electrochemical gradient (⌬p) resulting from the action of respiratory (or photosynthetic) electron transport. For recent reviews, see Refs. 1-3.The gross structure of transhydrogenases from different species is strikingly invariant. The enzyme has three components; dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude from the membrane (on the cytoplasmic side in bacteria, and the matrix side in mitochondria), and dII spans the membrane. Crystal structures of the dIII components of human and bovine transhydrogenases were recently described (4 -6), and the solution structure of the Rhodospirillum rubrum equivalent was determined by NMR.1 The basic fold of dIII is similar to the classical, dinucleotide-binding domain of lactate dehydrogenase, but NADP ϩ is bound with an unusual, "reversed" orientation. The nicotinamide ring of the bound NADP ϩ is exposed on a ridge of dIII. A homology model of dI (8), based on the crystal structure of the sequence-related alanine dehydrogenase (9), suggests that the nicotinamide ring of NADH is located in a deep cleft. It was proposed that, in the complete transhydrogenase, the ridge of dIII inserts into the cleft of dI to bring the nicotinamide rings of the two nucleotides into apposition to effect direct hydride transfer (5, 6). The protruding helix-D/loop-D of dIII is thought to interact with the membrane-spanning, dII and, together with the adjacent, lidlike loop E, which passes over the bound nucleotide, might be responsible for the energy transmission. The proton translocation steps during turnover are probably coupled specifically to changes in the mode of NADP(H) binding (1,5,10).Kinetic studies of transhydrogenase have also developed, following discoveries that fragments of the protein retain their nucleotide binding and some catalytic capacity. Thus, recombinant dI and dIII proteins, which bind their cognate nucleotides, have now been isolated from a number of species (11-16); mixtures of these proteins, even from different species, catalyze transhydrogenation reactions, albeit with properties that are modified relative to those of the complete enzyme. Transient state experiments, in particular, have revealed useful information on the hydride transfer step (17)(18)(19). Without exception, experiments with dI⅐dIII complexes have been carried out with nucleotide analogues having altered absorbance spectra to facilitate measurement of the rate of reaction. In this report we describe transient state experiments with the physiological nucleotides. A procedure is described, in which singl...

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Section: Figmentioning

confidence: 80%

Section: Single-turnover Transhydrogenation Revealed By Changes Inmentioning

confidence: 84%

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Venning

Peake²,

Quirk

et al. 2000

Journal of Biological Chemistry

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“…20–25 kDa and the use of deuteration to suppress relaxation has extended this range to 30 kDa and above. Recently, we [9] and others [10] used NMR techniques in the characterization of the NADP(H)‐binding domain from E. coli and R. rubrum , respectively, and a secondary structure model of the R. rubrum domain was also reported [11].…”

Section: Introductionmentioning

confidence: 99%

NMR characterization of the NADP(H)‐binding domain of Escherichia coli transhydrogenase: sequential assignment and global fold

et al. 1999

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The soluble NADP(H)-binding domain of Escherichia coli transhydrogenase (186 amino acids, 20.4 kDa, rotational correlation time 14 ns) was characterized using NMR techniques. The global fold is similar to that of a classical dinucleotidebinding fold with six parallel L L-strands in a central sheet surrounded by helices and irregular structures, but is lacking both K KD and K KE. The substrate is bound in an extended conformation at the C-terminal end of the parallel L L-sheet and our data support the notion of a redox dependent structural rearrangement.z 1999 Federation of European Biochemical Societies.

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“…Wild-type dI and wild-type dIII from R. rubrum transhydrogenase were expressed from the plasmids, pCD1 (23) and pNIC2 (24), respectively, in appropriate strains of Escherichia coli after induction with isopropyl ␤-D-thiogalactoside, as described in the earlier reports. The dI was purified on three consecutive chromatography columns, Q-Sepharose Fast Flow (GE Healthcare), Butyl Toyopearl (Tosohaas), and Q-Sepharose High Performance (GE Healthcare), essentially as described (23,25).…”

Section: Methodsmentioning

confidence: 99%

Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

Obiozo

Brondijk

White

et al. 2007

Journal of Biological Chemistry

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Transhydrogenase couples the redox reaction between NADH and NADP؉ to proton translocation across a membrane. The protein has three components: dI binds NADH, dIII binds NADP ؉ , and dII spans the membrane. Transhydrogenase is a "dimer" of two dI-dII-dIII "monomers"; x-ray structures suggested that the two catalytic sites alternate during turnover. Invariant Tyr 146 in recombinant dI of Rhodospirillum rubrum transhydrogenase was substituted with Phe and Ala (proteins designated dI.Y146F and dI.Y146A, respectively). Analytical ultracentrifuge experiments and differential scanning calorimetry show that dI.Y146A more readily dissociates into monomers than wild-type dI. Analytical ultracentrifuge and Trp fluorescence experiments indicate that the dI.Y146A monomers bind NADH much more weakly than dimers. Wild-type dI and dI.Y146F reconstituted activity to dI-depleted membranes with similar characteristics. However, dI.Y146A reconstituted activity in its dimeric form but not in its monomeric form, this despite monomers retaining their native fold and binding to the dI-depleted membranes. It is suggested that transhydrogenase reconstructed with monomers of dI.Y146A is catalytically compromised, at least partly as a consequence of the lowered affinity for NADH, and this results from lost interactions between the nucleotide binding site and the protein ␤-hairpin upon dissociation of the dI dimer. The importance of these interactions and their coupling to dI domain rotation in the mechanism of action of transhydrogenase is emphasized. Two peaks in the 1 H NMR spectrum of wild-type dI are broadened in dI.Y146A and are tentatively assigned to S-methyl groups of Met resonances in the ␤-hairpin, consistent with the segmental mobility of this feature in the structure.Transhydrogenase is located in the inner mitochondrial membrane of animal cells and in the cytoplasmic membrane of bacteria. It catalyzes the reduction of NADP ϩ by NADH coupled to the translocation of protons across the membrane shown in Reaction 1.The enzyme provides NADPH for biosynthesis reactions (1, 2) and for glutathione reduction in the protection of cells against oxidative stress (3-5). A role for transhydrogenase has also been implicated in glucose-stimulated insulin secretion by pancreatic ␤-cells (6).Transhydrogenase has three components. The dI component, which binds NADH, and the dIII component, which binds NADP ϩ , protrude from the membrane, and the dII component spans the membrane. Conformational changes link the redox reaction at the dI/dIII interface with proton translocation through dII (for recent reviews, see Refs. 7,8). The intact enzyme is a "dimer" of two dI-dII-dIII "monomers" (9 -12), although the disposition of dI, dII, and dIII along polypeptides is somewhat variable between species (13). Solution studies (14, 15) and x-ray structures (16 -20) reveal the profound asymmetry of a complex formed from mixtures of dI and dIII (the socalled dI 2 dIII 1 complex) of the Rhodospirillum rubrum enzyme. This was taken to suggest that transhydrogen...

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Untitled

Cited by 16 publications

References 4 publications

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

NMR characterization of the NADP(H)‐binding domain of Escherichia coli transhydrogenase: sequential assignment and global fold

Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

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