Proton-translocating nicotinamide nucleotide transhydrogenase of Escherichia coli. Involvement of aspartate 213 in the membrane-intercalating domain of the beta subunit in energy transduction.
Mutations in the beta subunit of Escherichia coli proton-translocating nicotinamide nucleotide transhydrogenase of the conserved residue beta Asp-213 to Asn (beta D213N) and Ile (beta D213I) resulted in the loss, respectively, of about 70% and 90% NADPH-->3-acetylpyridine adenine dinucleotide (AcPyAD) transhydrogenation and coupled proton translocation activities. However, the cyclic NADP(H)-dependent NADH-->AcPyAD transhydrogenase activities of the mutants were only approximately 35% inhibited. The latter transhydrogenation, which is not coupled to proton translocation, occurs apparently via NADP under conditions that enzyme-NADP(H) complex is stabilized. Mutations beta D213N and beta D213I also resulted in decreases in apparent KmNADPH for the NADPH-->AcPyAD and S0.5NADPH (NADPH concentration needed for half-maximal activity) for the cyclic NADH-->AcPyAD transhydrogenation reactions, and in KdNADPH, as determined by equilibrium binding studies on the purified wild-type and the beta D213I mutant enzymes. These results point to a structural role of beta Asp-213 in energy transduction and are discussed in relation to our previous suggestion that proton translocation coupled to NADPH-->NAD (or AcPyAD) transhydrogenation is driven mainly by the difference in the binding energies of NADPH and NADP.
Yamaguchi
M
M
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037, USA.
Hatefi
Y
Y
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
D009243:NAD; D009249:NADP; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases