Mutation of the conserved amino acids of mitochondria aldehyde dehydrogenase. Role of the conserved residues in the mechanism of reaction.

Abstract

During the last three decades much have been accomplished to help us understand the mechanism of aldehyde dehydrogenase. Earlier chemical modification studies suggested that C302 and E268 could be essential residues. We used site directed mutagenesis to elucidate the importance of these residues in catalysis. The enzyme was completely inactive when C302 was replaced by alanine. However, replacing C302 by serine did produce a mutant with some catalytic activity. This led us to conclude the C302 may function as a nucleophile (Farres et al., 1995). Glutamate at position 268 was shown to act as a general base to activate the nucleophile that led to the initiation of the catalytic reaction (Wang and Weiner, 1995). There were other residues such as C49, S74, C162 and H235 which were thought to be essential based on the inhibition and protection experiments (Weiner et al., 1985; Tu and Weiner, 1988; Loomes et al., 1990). Later mutational analysis showed that replacing them with other residues did not render the enzyme completely inactive, hence, they might not be critical for the functioning of the enzyme (Zheng and Weiner, 1993; Rout and Weiner, 1994; Farres et al., 1995).