Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli. Effects on catalytic and H+-pumping activities
| Autor: | Magnus A B Axelsson, Tatyana V. Korneenko, Ola Fjellström, Maria V. Ivanova, Nikolay B. Pestov, Michail Shakhparonov, Maxim V Egorov, Tania Bizouarn, Jan Rydström |
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| Rok vydání: | 2000 |
| Předmět: |
Models
Molecular Protein Denaturation Protein Conformation Recombinant Fusion Proteins Detergents Molecular Sequence Data medicine.disease_cause Rhodospirillum rubrum Biochemistry Catalysis Mice Protein structure Bacterial Proteins medicine Escherichia coli NADP Transhydrogenases Animals Trypsin Amino Acid Sequence Peptide sequence G alpha subunit chemistry.chemical_classification Mice Inbred BALB C biology Antibodies Monoclonal Hydrogen-Ion Concentration biology.organism_classification NAD Antibodies Bacterial Protein Structure Tertiary Transmembrane domain Enzyme chemistry Liposomes NAD+ kinase Protein Multimerization Protons NADP Protein Binding |
| Zdroj: | European journal of biochemistry. 267(11) |
| ISSN: | 0014-2956 |
| Popis: | Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the alpha and the beta subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II. The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E. coli was investigated using the separately expressed and purified domain I from R. rubrum, and His-tagged intact and trypsin-treated E. coli transhydrogenase. Despite harsh treatments with, e.g. detergents and denaturing agents, the alpha and beta subunits remained tightly associated. A monoclonal antibody directed towards the alpha subunit was strongly inhibitory, an effect that was relieved by added rrI. In addition, rrI also reactivated the trypsin-digested E. coli enzyme in which domain I had been partly removed. This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II. Replacement of domain I of intact, as well as trypsin-digested, E. coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction. The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions. |
| Databáze: | OpenAIRE |
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