Identification of an aspartic acid residue in the beta subunit which is essential for catalysis and proton pumping by transhydrogenase from Escherichia coli.
Based on the alignment of 7 unknown amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highly conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H)-binding site of the beta subunit. The effect of replacing the four conserved acidic residues, betaE361, betaE374, betaD383 and betaD392, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, betaE371, which is not conserved but located in the same domain, was also mutated. Of these residues, betaAsp 392 proved to be the only residue which is essential for both activities. However, two betaAsp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADPH, suggesting that the mutations did not cause a global change but rather a subtle local change influencing the dissociation of NADP(H). It is proposed that betaAsp 392 together with th previously identified betaHis91 form part of a proton wire in transhydrogenase.
Meuller
J
J
Department of Biochemistry, Lundberg Labortary, Goteborg University, Sweden.
Hu
X
X
Bunthof
C
C
Olausson
T
T
Rydström
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D046911:Macromolecular Substances; D001224:Aspartic Acid; D009249:NADP; D009250:NADP Transhydrogenases