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, R., and Lindahl, R. (1993) Protein Sci, 2, 1890-1900). Of these 14 were glycines and prolines, Site-directed mutagenesis showed that Cys(302) was the essential nucleophile and that Glu(268) was the general base necessary to activate Cys(302) for both the dehydrogenase and esterase reaction, Here we report the mutational analysis of other conserved residues possessing reactive side chains Arg(84), Lys(192), Thr(384), Glu(399), and Ser(471), along with partially conserved Glu(398) and Lys(489), to determine their involvement in the catalytic process and correlate these finding with the known structure of mitochondrial ALDH (Steinnetz, 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 K-m for aldehyde significantly, although k(3), the rate constant calculated for aldehyde binding was decreased, The K-m and dissociation constant (R-ia) for NAD(+) increased significantly for R192Q and S471A compared with the native enzyme, Mutations of only Lys(192) and Glu(399), 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.