Revised Manuscript Received December 1, 1994® abstract: To determine the active site cysteine residue in aldehyde dehydrogenase, we mutated amino acid residues 49, 162, and 302 of recombinantly expressed rat liver mitochondrial (class 2) aldehyde dehydrogenase. The C49A and C162A mutants were hilly active tetrameric enzymes, although the Cl62A 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 nucleophile, the residue was mutated to a serine, a poor nucleophile. This C302Smutant was active but was a much poorer catalyst, with a kcJKm value 7 x 105 times lower than that of the recombinant native enzyme. Unlike with native enzyme where deacylation is rate limiting, formation of the serine hemiacetal intermediate appearedto be the rate-limiting step. Cysteine 302 is the only strictly conserved cysteine residue among all the available sequences of the aldehyde dehydrogenase superfamily, supporting the role of this residue as the active site nucleophile of aldehyde dehydrogenase.

NAD (P)+-dependent aldehyde dehydrogenases (ALDH) 1 (EC 1.2. 1.3) catalyze the dehydrogenation of aldehydes, as well as the hydrolysis of activated esters. The aldehyde dehydrogenase superfamily includes broad substrate en-zymes, such as class 1 (cytosolic), class 2 (mitochondrial), class 3 (tumor-associated), and microsomal aldehyde dehy-