The potential roles of the conserved amino acids in human liver mitochondrial aldehyde dehydrogenase.
The sequence alignment of all known aldehyde dehydrogenases showed that only 23 residues were completely conserved (Hempel, J., Nicholas, H., and Lindahl, R. (1993) Protein Sci. 2, 1890-1900). Of these 14 were glycines and prolines. Site-directed mutagenesis showed that Cys302 was the essential nucleophile and that Glu268 was the general base necessary to activate Cys302 for both the dehydrogenase and esterase reaction. Here we report the mutational analysis of other conserved residues possessing reactive side chains Arg84, Lys192, Thr384, Glu399, and Ser471, along with partially conserved Glu398 and Lys489, to determine their involvement in the catalytic process and correlate these finding with the known structure of mitochondrial ALDH (Steinmetz, C. G., Xie, P.-G., Weiner, H., and Hurley, T. D. (1997) Structure 5, 701-711). No residue was found to be absolutely essential, but all the mutations caused a decrease in the specific activity of the enzyme. None of the mutations affected the Km for aldehyde significantly, although k3, the rate constant calculated for aldehyde binding was decreased. The Km and dissociation constant (Kia) for NAD+ increased significantly for K192Q and S471A compared with the native enzyme. Mutations of only Lys192 and Glu399, both NAD+-ribose binding residues, led to a change in the rate-limiting step such that hydride transfer became rate-limiting, not deacylation. Esterase activity of all mutants decreased even though mutations affected different catalytic steps in the dehydrogenase reaction.
Sheikh
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
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D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D017124:Conserved Sequence; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D016415:Sequence Alignment
D000596:Amino Acids; D005973:Glutamine; D009243:NAD; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D008239:Lysine