Kinetic resolution of the reaction catalysed by proton‐translocating transhydrogenase from <i>Escherichia coli</i> as revealed by experiments with analogues of the nucleotide substrates

Hutton, Mike; Day, Joanna M.; Bizouarn, Tania; Jackson, John L.

doi:10.1111/j.1432-1033.1994.tb18587.x

Cited by 85 publications

(90 citation statements)

References 39 publications

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“…Previous experiments established that nucleotide binding to domain I is also pH independent [11], and is therefore unlikely to be coupled to proton pumping. In fact, it is becoming increasingly likely that the proton-translocation reactions of transhydrogenase are centred on the NADP ϩ /NADPH binding/release reactions of domain III [1,28,29]. As emphasised above, the release of product NADP ϩ from domain III is extremely slow in the absence of domain II, and this suggests that, in the complete enzyme, an interaction between domain II and the two peripheral domains is responsible for accelerating the release of the nucleotide, and therefore the steady-state rate of transhydrogenation [9].…”

Section: Discussionmentioning

confidence: 99%

Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Venning¹,

Bizouarn²,

Cotton³

et al. 1998

European Journal of Biochemistry

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Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD ϩ /NADH, and domain III for NADP ϩ /NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD ϩ analogue, acetylpyridine adenine dinucleotide (AcPdAD ϩ ). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP ϩ (kϷ0.03 s Ϫ1 ). Phase A of the burst (kϷ600 s Ϫ1 ) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (kϷ10Ϫ50 s Ϫ1 ), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or Keywords : transhydrogenase; stopped flow; proton translocation; recombinant protein; membrane protein.Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across a membrane (for reviews,(1) The enzyme is found in animal mitochondria and in bacteria. Probably, under most physiological conditions, the reaction is driven to the right, towards NADPH formation, by the proton motive force generated by the respiratory (or photosynthetic) electron-transport chain.Transhydrogenase has three domains. Domains I and III protrude from the membrane and possess the nucleotide-binding sites ; domain I for NAD ϩ and NADH, and domain III for NADP ϩ and NADPH [1Ϫ3]. Domain II spans the membrane and might comprise 10Ϫ12 transmembrane helices [4].Recombinant forms of domain I from Rhodospirillum rubrum [5,6] and Escherichia coli [7,8] and purified. The isolated domains bind their respective nucleotides with high specificity and affinity [5Ϫ10, 11]. A mixture of the two domains from the R. rubrum protein catalyses transhydrogenation, even in the absence of the membrane-spanning domain II [6,9], but the rates of the 'forward' and 'reverse' transhydrogenation reactions catalysed by the mixture are very slow Ϫ they are profoundly limited by release of product NADPH (k off Ϸ 5ϫ10 Ϫ4 s Ϫ1 ) and NADP ϩ (k off Ϸ 0.03 s Ϫ1 ), respectively, from domain III [9]. Evidently, in the complete enzyme, release of NADP(H) is accelerated by domain II. The mixture of domains I and III...

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Section: Discussionmentioning

confidence: 99%

Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Venning¹,

Bizouarn²,

Cotton³

et al. 1998

European Journal of Biochemistry

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“…X-ray structures reveal nucleotidebinding conformations in which the block may be achieved; essentially the nicotinamide and dihydronicotinamide rings are held apart by the enzyme [24]. The pH dependences of transhydrogenation reactions in bacterial membranes suggest that protonation of the enzyme converts open dIII into its occluded state and, subsequent to the hydride transfer step, deprotonation then converts occluded dIII back into its open state [51,54,55]. Thus, the basic mechanism is (i) substrate nucleotides, NADP + and NADH, associate with their binding sites in an enzyme protomer having both dIII and dI in open states, (ii) protonation of dII from the outside aqueous phase converts the open dIII to occluded, (iii) hydride-ion equivalents are transferred from bound NADH to NADP + , (iv) deprotonation of dII on the cytoplasmic side regenerates the open state of dIII, and (v) the nucleotide products, NADPH and NAD + , are released.…”

Section: The Binding-change Mechanism Of Coupling To Proton Translocamentioning

confidence: 99%

“…This takes place with the dIII component in an ''occluded state'' in which both the binding and release of NADP + and NADPH are extremely slow relative to enzyme turnover [51][52][53]. Crystal structures show that with dIII in the occluded state the pro-R hydrogen atom on C4 of the dihydronicotinamide ring of NADH (on dI) can be brought into close apposition with the si face of C4 of the nicotinamide ring of NADP + (on dIII) to effect direct, stereo-specific and rapid hydride transfer [21].…”

Section: The Binding-change Mechanism Of Coupling To Proton Translocamentioning

confidence: 99%

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

et al. 2015

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Edited by Peter BrzezinskiKeywords: Transhydrogenase Membrane-protein structure Nicotinamide nucleotide Proton-pump Proton-gating a b s t r a c tThe membrane protein transhydrogenase in animal mitochondria and bacteria couples reduction of NADP + by NADH to proton translocation. Recent X-ray data on Thermus thermophilus transhydrogenase indicate a significant difference in the orientations of the two dIII components of the enzyme dimer (Leung et al., 2015). The character of the orientation change, and a review of information on the kinetics and thermodynamics of transhydrogenase, indicate that dIII swivelling might assist in the control of proton gating by the redox state of bound NADP + /NADPH during enzyme turnover.

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“…We have described a detailed study of the cyclic reduction of AcPdAD' by NADH in the presence of either NADP' or NADPH by the complete detergent-dispersed transhydrogenase purified from E. coli [20,211. The reaction was very fast (approximately 50 times the rate of the simple reverse reaction at pH 6.0) and depended crucially on the tight binding of NADP' or NADPH to the enzyme.…”

Section: Optical Properties Of the Domain 111 Proteinmentioning

confidence: 99%

“…It proceeds through the reduction of NADP' by NADH, and the subsequent re-oxidation of NADPH by AcPdAD', while NADP'NADPH remain bound to the enzyme [20,211. It was shown from the analysis of the pH dependences of transhydrogenation reactions of the detergentsolubilized E. coli enzyme that the release of NADP' and NADPH are accompanied by the release of a proton [20, 211. There is good reason to believe that the protolytic reactions accompanying the binding and release of the NADP(H) are components of hydrogen ion translocation in the membrane-bound enzyme, and this has provided the basis for a working model to explain the proton-pumping action of transhydrogenase [20,21 1. For the E. coli enzyme at low pH, it was argued that the rates of simple forward (reduction of thio-NADP' by NADH) and reverse (reduction of AcPdAD' by NADPH) transhydrogenation are limited by release from the enzyme of thio-NADPH and NADP', respectively [20,211. The cyclic reaction, however, relies on the presence of tightly bound NADP(H); deuterium isotope measurements showed that its rate is limited by the H--transfer steps.…”

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confidence: 99%

Properties of the Purified, Recombinant, NADP(H)‐Binding Domain III of the Proton‐Translocating Nicotinamide Nucleotide Transhydrogenase from Rhodospirillum Rubrum

Diggle

Bizouarn

Cotton

et al. 1996

European Journal of Biochemistry

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Transhydrogenase comprises three domains. Domains I and I11 are peripheral to the membrane and possess the NAD(H)-and NADP(H)-binding sites, respectively, and domain 11 spans the membrane. Domain TIT of transhydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli, and purified. The purified protein was associated with substoichiometric quantities of tightly bound NADP' and NADPH. Fluorescence spectra of the domain 111 protein revealed emissions due to Tyr residues. Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nucleotide are spatially close. The rate constants for NADP' release and NADPH release from domain Ill were 0.03 SKI and S.6X10-4s-1, respectively. In the absence of domain I1 a mixture of the recombinant domain 111 protein, plus the previously described recombinant domain 1 protein, catalysed reduction of acetylpyridine-adenine dinucleotide (AcPdAD') by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP' from domain 111. Similarly, the mixture catalysed reduction of thio-NADP' by NADH (forward transhydrogenation) at a rate limited by release of thio-NADPH from domain 111. The mixture also catalysed very rapid reduction of AcPdAD' by NADH, probably by way of a cyclic reaction mediated by the tightly bound NADP(H). Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain 111 and vice versa indicated (a) that during reduction of AcPdAD' by NADPH, a single domain I protein can visit and transfer H ~ equivalents to about 60 domain 111 proteins during the time taken for a single domain I11 to release its NADP ' , whereas (b) the cyclic reaction is rapid on the timescale of formation and breakdown of the domain I . 111 complex. The rate of the hydride transfer reaction was similar in thc domain I I I11 complex to that in the complete membrane-bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I . 111 complex due to the greatly decreased rates of release of NADP' and NADPH. It is concluded that, in the complete enzyme, conforniational changes in the membrane-spanning domain 11, which result from proton translocation, lead to changes in the binding affinity of domain 111 for NADP' and for NADPH.

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Kinetic resolution of the reaction catalysed by proton‐translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates

Cited by 85 publications

References 39 publications

Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

Properties of the Purified, Recombinant, NADP(H)‐Binding Domain III of the Proton‐Translocating Nicotinamide Nucleotide Transhydrogenase from Rhodospirillum Rubrum

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