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.
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.
Bizouarn
T
T
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Fjellström
O
O
Axelsson
M
M
Korneenko
T V
TV
Pestov
N B
NB
Ivanova
M V
MV
Egorov
M V
MV
Shakhparonov
M
M
Rydström
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002384:Catalysis; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D003902:Detergents; D008081:Liposomes; D011522:Protons; D011993:Recombinant Fusion Proteins; D009243:NAD; D009249:NADP; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases; D014357:Trypsin