Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

Yamaguchi, Mutsuo; Hatefi, Youssef

doi:10.1021/bi00440a049

Cited by 25 publications

(8 citation statements)

References 24 publications

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“…Further, the B-face is more exposed to solvent, consistent with biochemical data requiring direct hydride ion transfer between the 4A position of NAD(H) and the 4B position of NADP(H) 1,2 . Chemical modification 10,11 and site-directed mutagenesis data [12][13][14][15][16][17] are in agreement with the observed binding of NADP. Based on these mutagenesis studies, and homology of the E. coli NADP(H) binding domain with classical nucleotide binding folds, a model has been proposed for the structure of the E. coli domain with bound NADP(H) 17 .…”

Section: Non-classical Binding Of Nadpsupporting

confidence: 81%

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et al. 1999

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The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 A resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.

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Section: Non-classical Binding Of Nadpsupporting

confidence: 81%

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et al. 1999

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“…The modification of cysteine residues in transhydrogenases has been studied both with the E. coli enzyme [29,30] and with the bovine enzyme [33][34][35][36]. Comparisons are difficult to make, since the residue corresponding to βCys-260 in E. coli transhydrogenase (residue 834 in the bovine enzyme) is the only conserved cysteine residue located in a domain composed of a sequence of about 12 conserved residues.…”

Section: Discussionmentioning

confidence: 99%

Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase

Meuller

Zhang

Hou

et al. 1997

Biochemical Journal

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Nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated with respect to the roles of its cysteine residues. This enzyme contains seven cysteines, of which five are located in the alpha subunit and two are in the beta subunit. All cysteines were replaced by site-directed mutagenesis. The final construct (alphaC292T, alphaC339T, alphaC395S, alphaC397T, alphaC435S, betaC147S, betaC260S) was inserted normally in the membrane and underwent the normal NADPH-dependent conformational change of the beta subunit to a trypsin-sensitive state. Reduction of NADP+ by NADH driven by ATP hydrolysis or respiration was between 32% and 65% of the corresponding wild-type activities. Likewise, the catalytic and proton pumping activities of the purified cysteine-free enzyme were at least 30% of the purified wild-type enzyme activities. The H+/H- ratio for both enzymes was 0.5, although the cysteine-free enzyme appeared to be more stable than the wild-type enzyme in proteoliposomes. No bound NADP(H) was detected in the enzymes. Modification of transhydrogenase by diethyl pyrocarbonate and the subsequent inhibition of the enzyme were unaffected by removal of the cysteines, indicating a lack of involvement of cysteines in this process. Replacement of cysteine residues in the alpha subunit resulted in no or little change in activity, suggesting that the basis for the decreased activity was probably the modification of the conserved beta-subunit residue Cys-260 or (less likely) the non-conserved beta-subunit residue Cys-147. It is concluded that the cysteine-free transhydrogenase is structurally and mechanistically very similar to the wild-type enzyme, with minor modifications of the properties of the NADP(H) site, possibly mediated by the betaC260S mutation. The cysteine-free construct will be a valuable tool for studying structure-function relationships of transhydrogenases.

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“…Specific activity (mol AcPyAD or AcPyADP reduced/min/mg protein) is expressed per mg of bovine or R. rubrum domain I where one or the other was present. The NADPH 3 AcPyAD activity of purified bovine transhydrogenase is 30 -32 mol AcPyAD reduced (min ⅐ mg) Ϫ1 (Yamaguchi and Hatefi, 1993 chi and Hatefi, 1989). As seen in Table II, NEM had no inhibitory effect on the R. rubrum ␣1 subunit but inhibited the bovine domain III.…”

Section: Table Imentioning

confidence: 96%

“…Also, similar to the intact bovine enzyme, addition of NADPH but not NADH stimulated the NEM inhibition. In the bovine transhydrogenase, it was shown that the reason for this augmentation of the NEM inactivation rate is that in the presence of NADPH, the effective pK a of Cys 893 is lowered from 9.1 to 8.7 (Yamaguchi and Hatefi, 1989). Not shown in Table II is the inhibitory effect of palmitoyl-CoA (Rydström, 1972).…”

Section: Table Imentioning

confidence: 99%

Proton-translocating Nicotinamide Nucleotide Transhydrogenase

Yamaguchi¹,

Hatefi²

1995

Journal of Biological Chemistry

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The nicotinamide nucleotide transhydrogenase of bovine mitochondria is a homodimer of monomer M(r) = 109,065. The monomer is composed of three domains, an NH2-terminal 430-residue-long hydrophilic domain I that binds NAD(H), a central 400-residue-long hydrophobic domain II that is largely membrane intercalated and carries the enzyme's proton channel, and a COOH-terminal 200-residue-long hydrophilic domain III that binds NADP(H). Domains I and III protrude into the mitochondrial matrix, where they presumably come together to form the enzyme's catalytic site. The two-subunit transhydrogenase of Escherichia coli and the three-subunit transhydrogenase of Rhodospirillum rubrum have each the same overall tridomain hydropathy profile as the bovine enzyme. Domain I of the R. rubrum enzyme (the alpha 1 subunit) is water soluble and easily removed from the chromatophore membranes. We have isolated domain I of the bovine transhydrogenase after controlled trypsinolysis of the purified enzyme and have expressed in E. coli and purified therefrom domain III of this enzyme. This paper shows that an active bidomain transhydrogenase lacking domain II can be reconstituted by the combination of purified bovine domains I plus III or R. rubrum domain I plus bovine domain III.

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Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

Cited by 25 publications

References 24 publications

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Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase

Proton-translocating Nicotinamide Nucleotide Transhydrogenase

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