INVESTIGATION OF THE ACTIVE-SITE CYSTEINE RESIDUE OF RAT-LIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE BY SITE-DIRECTED MUTAGENESIS

To determine the active site cysteine residue in aldehyde dehydrogenas e, we mutated amino acid residues 49, 162, and 302 of recombinantly ex pressed rat liver mitochondrial (class 2) aldehyde dehydrogenase. The C49A and C162A mutants were fully active tetrameric enzymes, although the C162A mutant was found to be highly unstable. The C302A mutant was also a tetramer and bound coenzyme, but lacked both dehydrogenase and esterase activities. To test for the role of cysteine 302 as a nucleo phile, the residue was mutated to a serine, a poor nucleophile. This C 302S mutant was active but was a much poorer catalyst, with a k(cat)/K -m value 7 x 10(5) times lower than that of the recombinant native enz yme. Unlike with native enzyme where deacylation is rate limiting, for mation of the serine hemiacetal intermediate appeared to be the rate-l imiting step. Cysteine 302 is the only strictly conserved cysteine res idue among all the available sequences of the aldehyde dehydrogenase s uperfamily, supporting the role of this residue as the active site nuc leophile of aldehyde dehydrogenase.