Mutation of conserved polar residues in the transmembrane domain of the proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli.
The pyridine nucleotide transhydrogenase carries out transmembrane proton translocation coupled to transfer of a hydride ion equivalent between NAD+ and NADP+. Previous workers (E. Holmberg et al. Biochemistry 33, 7691-7700, 1994; N. A. Glavas et al. Biochemistry 34, 7694-7702, 1995) had examined the role in proton translocation of conserved charged residues in the transmembrane domain. This study was extended to examine the role of conserved polar residues of the transmembrane domain. Site-directed mutagenesis of these residues did not produce major effects on hydride transfer or proton translocation activities except in the case of betaAsn222. Most mutants of this residue were drastically impaired in these activities. Three phenotypes were recognized. In betaN222C both activities were impaired maximally by 70%. The retention of proton translocation indicated that betaAsn222 was not directly involved in proton translocation. In betaN222H both activities were drastically reduced. Binding of NADP+ but not of NADPH was impaired. In betaN222R, by contrast, NADP+ remained tightly bound to the mutant transhydrogenase. It is concluded that betaAsn222, located in a transmembrane alpha-helix, is part of the conformational pathway by which NADP(H) binding, which occurs outside of the transmembrane domain, is coupled to proton translocation. Some nonconserved or semiconserved polar residues of the transmembrane domain were also examined by site-directed mutagenesis. Interaction of betaGlu124 with the proton translocation pathway is proposed.
Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada.
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006865:Hydrogenation; D007425:Intracellular Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017494:Proton Pumps / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D017494:Proton Pumps; D009243:NAD; D001216:Asparagine; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases