Evidence for reactivity of serine-74 with trans-4-(N,N-dimethylamino)cinnamaldehyde during oxidation by the cytoplasmic aldehyde dehydrogenase from sheep liver

Abstract

A nucleophilic group in the active site of aldehyde dehydrogenase, which covalently binds the aldehyde moiety during the enzyme-catalyzed oxidation of aldehydes to acids, was acylated with the chromophoric aldehyde trans-4-(N,N-dimethylamino)cinnamaldehyde (DACA). Acyl-enzyme trapped by precipitation with perchloric acid was digested with trypsin, and the peptide associated with the chromophoric group was isolated and shown to be Gln-Ala-Phe-Gln-Ile-Gly-Ser-Pro-Trp-Arg. After redigestion with thermolysin, the chromophore was associated with the C-terminal hexaresidue part. If the chromophore is attached to this peptide, serine would be expected to bind the aldehyde and lead to the required acylated derivative. Differential labeling experiments were performed in which all free thiol groups on the acylated enzyme were blocked by carboxymethylation. The acyl chromophore was then removed by controlled hydrolysis and the protein reacted with [14C]iodoacetamide. No 14C-labeled tryptic peptides were isolated, suggesting that the sulfur of a cysteine cannot be the acylated residue in the precipitated acyl-enzyme.

Involvement of Glutamate 268 in the Active Site of Human Liver Mitochondrial (Class 2) Aldehyde Dehydrogenase As Probed by Site-Directed Mutagenesis

“…That is, an active site nucleophile attacks the aldehyde to form a thiohemiacetal intermediate, which is then oxidized to form an acyl enzyme. Site-directed mutagenesis analysis verified the earlier conclusion that cysteine 302 is the active site nucleophile (FarrCs et al, 1995) and not serine 74 (Rout & Weiner, 1994) as proposed by Loomes et al (1990) or cysteine 49 as we concluded from chemical modification studies (Tu & Weiner, 1988a). After the thioacyl intermediate is formed, it must be hydrolyzed, presumably by the action of general base catalysis.…”

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

“…By performing site-directed mutagenesis with the rat liver enzyme, we now find that the residue is cysteine 302 (63), as originally suggested by Hempel and Pietruszko (25). Recently, however, it was found that in sheep liver cytosolic ALDH, serine 74 could be modified by a substrate (38 It was reported that glutamate 268 in the human cytosolic enzyme appeared to be essential for activity (1,2). This is also a highly conserved residue in all species.…”

Functional characterization of a Drosophila melanogaster succinic semialdehyde dehydrogenase and a non-specific aldehyde dehydrogenase

“…Furthermore, it was surmised that one or more amino acid residues capable of acid base catalysis (the 'general base') were involved in the hydrolytic cleavage of the postulated thioacyl intermediate formed by hydride transfer to the NAD(P) + coenzyme (Jakoby, 1958). The identities of the thiohemiacetal-forming cysteine residue and the general base were investigated by biochemical techniques, initially with in part controversial results (Hempel and Pietrusko, 1981;Hempel et al, 1985;Tu and Weiner, 1988;Abriola et al, 1987Abriola et al, , 1990Loomes et al, 1990;Blatter et al, 1990;Kitson et al, 1991). Finally, their identities and positions were established in mammalian mitochondrial ALDH by a series of site-directed mutagenesis experiments (Zheng and Weiner, 1993;Farres et al, 1995;Wang and Weiner, 1995) as cysteine 305 (C305) and glutamate 268 (E268), respectively.…”