Probing the Active Site of Aldehyde Dehydrogenase by Site Directed Mutagenesis
It has generally been accepted that aldehyde dehydrogenase functions through covalent catalysis where a nucleophilic amino acid residue attacks the carbonyl of the aldehyde substrate (Weiner et al., 1982). Since the first purification on ALDH by Feldman and Weiner in 1972, it has been argued that the active site nucleophile was a cysteine residue. The identity of this specific cysteine in the primary structure of the enzyme however, has not been unequivocally determined. Recently it has been suggested that the nucleophile is not cysteine but is serine (Loomis et al., 1990). A similar adduct could be formed with either amino acid; a thiohemiacetal in the former, a hemiacetal in the latter. The intermediate would then be oxidized in the presence of NAD to produce a thioacyl or an acyl intermediate respectively, as illustrated in Fig. 1.
KeywordsSite Directed Mutagenesis Aldehyde Dehydrogenase Active Site Residue Pulse Chase Experiment Horse Liver
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