PMID:11502193
| Citation |
Althage, M, Bizouarn, T and Rydström, J (2001) Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E. coli transhydrogenase. Biochemistry 40:9968-76 |
|---|---|
| Abstract |
The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport. In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266. A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values. It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III. |
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| Keywords |
Amino Acid Sequence; Anilino Naphthalenesulfonates/chemistry; Anilino Naphthalenesulfonates/metabolism; Cell Membrane/enzymology; Cysteine/chemistry; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Hydrogen-Ion Concentration; Kinetics; Magnesium Chloride/pharmacology; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; NADP/chemistry; NADP/metabolism; NADP Transhydrogenases/chemistry; Plasmids/metabolism; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Proteolipids/metabolism; Protons; Spectrometry, Fluorescence; Time Factors |
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