Interaction of purified nicotinamide nucleotide transhydrogenase with dicyclohexylcarbodiimide

Phelps, Donna C.; Hatefi, Youssef

doi:10.1021/bi00314a037

Cited by 59 publications

(42 citation statements)

References 17 publications

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“…Because hydride transfer across the single dI/dIII interface of the complex is extremely rapid (25), it was proposed that in the complete enzyme the two dI/dIII interfaces must be alternately brought together during turnover. The proposal is supported by earlier studies indicating "half of the sites" reactivity for bovine transhydrogenase (26,27) and by another investigation in which inactivation of the bovine enzyme by high concentrations of Triton X-100 was attributed to the dissociation of dimers into monomers (18).…”

supporting

confidence: 53%

The Membrane-peripheral Subunits of Transhydrogenase fromEntamoeba histolytica Are Functional Only When Dimerized

Weston¹,

Venning

Jackson

2002

Journal of Biological Chemistry

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Unlike their bacterial and mammalian counterparts, the NADP(H)-and NAD(H)-binding components of proton-translocating transhydrogenase from the protozoan parasite Entamoeba histolytica (denoted ehdIII and ehdI, respectively) are tethered by a polypeptide linker. The recombinant tethered fragment, ehdIII-ehdI, was prepared without its membrane-spanning dII component. Dimers of ehdIII-ehdI catalyzed transhydrogenation, but monomers were inactive. The addition of ehdIII to ehdIIIehdI monomers did not lead to an increase in the rate of transhydrogenation, showing that this inactivity is not the result of an unfavorable topology introduced by the linker. The addition of a bacterial dI to ehdIII-ehdI led to an increase in the rate of transhydrogenation, showing that the linker is flexible. A hybrid protein in which ehdIII is tethered to the bacterial dI (denoted ehdIII-rrdI) more readily formed active dimers. Data from small angle x-ray scattering by the hybrid dimers were fitted to models derived from the high-resolution crystal structure of the bacterial dI 2 Some membrane proteins, through changes in their conformation, can link a scalar chemical reaction to the translocation of solutes or ions. Often in these proteins, oligomeric interactions are central to the mechanism. For example, the F 1 F oATPase operates by a 3-site rotary mechanism (1) and the P-glycoprotein undergoes a 2-site alternation during turnover (2). Although it catalyzes a redox reaction rather than ATP hydrolysis, transhydrogenase is in this category of proteins, and its amenable properties make it an excellent model for studying conformational coupling (reviewed in Refs. 3 and 4). The enzyme links the hydride transfer between NAD(H) and NADP(H) to the translocation of protons across the inner membranes of mammalian mitochondria and the cytoplasmic membranes of bacteria as shown in Reaction 1.The reaction is driven from left to right by the proton electrochemical gradient generated by the respiratory (or sometimes photosynthetic) electron transport chain. Thus, transhydrogenase is important as a source of NADPH for biosynthesis and glutathione reduction (for protection against free-radical damage) and in the regulation of flux through the tricarboxylic acid cycle (5, 6). Genes encoding transhydrogenase have also been found in several protozoan parasites. In Plasmodium falciparum the enzyme is probably located in the mitochondrial membrane, but in Eimeria tenella it is thought to be associated with so-called "refractile bodies" (7) and in Entamoeba histolytica, with "mitosomes" (8), otherwise known as "cryptons" (9). The functions of the Ei. tenella refractile bodies and the En. histolytica mitosomes and the nature of the partner proteins that are coupled to the transhydrogenase in the local chemiosmotic proton circuits are not known. The partner proteins are probably not enzymes normally associated with oxidative phosphorylation (10). All proton-translocating transhydrogenases seem to have a similar structural organization. There is a dII compon...

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supporting

confidence: 53%

The Membrane-peripheral Subunits of Transhydrogenase fromEntamoeba histolytica Are Functional Only When Dimerized

Weston¹,

Venning

Jackson

2002

Journal of Biological Chemistry

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Section: Reactionsupporting

confidence: 68%

“…This was taken to suggest that transhydrogenase operates by an "alternating-sites" mechanism in which the two dIdII-dIII monomers run 180 o out-of-phase during catalytic turnover. Consistent with this, earlier work had indicated halfof-the-sites inhibition of bovine transhydrogenase by covalent modifiers (21,22).The alternating sites mechanism requires that events in the two dI-dII-dIII monomers are coordinated; there must be a coupling of conformational changes across the dimer interface. In this report, we describe the effects of substituting invariant Tyr 146 , a residue which is centrally located in the dimer interface between the two dI components of R. rubrum transhydrogenase (see Fig.…”

mentioning

confidence: 63%

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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“…Phelps and Hatefi [26] have suggested that (CHXN)~C reacts near the NAD(H) binding site of beef heart mitochondria1 transhydrogenase. By contrast, Persson et al [18] observed an inhibition of proton pump activity without an effect on uncoupled catalytic activity, suggesting that proton translocation and catalytic activities are not obligatorily linked.…”

Section: -mentioning

confidence: 99%

Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli

Clarke

Bragg

1985

European Journal of Biochemistry

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1. The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, CI and j, of apparent M , 50000 and 47000.2. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine.3. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [ ''C]Dicyclohexylcarbodiimide preferentially labelled the 50 000-M, subunit of the transhydrogenase enzyme.4. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.Pyridine nucleotide transhydrogenase, found in the cytoplasmic membrane of Escherichia coli and in the inner membrane of mitochondria, catalyzes the reversible transfer of a hydride ion equivalent between NAD and NADP. The transhydrogenation between NADH and NADP is coupled to respiration and ATP hydrolysis (see [I] for review).During the past decade, a large body of evidence has accumulated from studies on the mitochondria1 transhydrogenase to support Mitchell's hypothesis [2] that the transhydrogenase functions as a proton pump and translocates protons across the membrane according to the equation [31: nH; + NADPH + NAD nH&, + NADP + NADH .Comparatively little work has been done with the corresponding enzyme from E. coli despite advantages that genetic manipulation of this system can offer. The elucidation of the subunit composition and molecular mechanism of the E. coli transhydrogenase requires purification and reconstitution of the functional enzyme. So far the E. coli transhydrogenase has been only partially purified [4-61. SDS/ polyacrylamide gels of the preparation purified by Liang andCorrespondence to

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Interaction of purified nicotinamide nucleotide transhydrogenase with dicyclohexylcarbodiimide

Cited by 59 publications

References 17 publications

The Membrane-peripheral Subunits of Transhydrogenase fromEntamoeba histolytica Are Functional Only When Dimerized

The Membrane-peripheral Subunits of Transhydrogenase fromEntamoeba histolytica Are Functional Only When Dimerized

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

Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli

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