Investigation of the Active Site Cysteine Residue of Rat Liver Mitochondrial Aldehyde Dehydrogenase by Site-Directed Mutagenesis

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 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 nucleophile, the residue was mutated to a serine, a poor nucleophile. this C302S mutant was active but was a much poorer catalyst, with a kcat/Km value 7 x 10(5) times lower than that of the recombinant native enzyme. Unlike with native enzyme where deacylation is rate limiting, formation of the serine hemiacetal intermediate appeared to 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.

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

“…The acylation step involves the formation of a hemithioacetal intermediate via the nucleophilic attack of the catalytic Cys-302 (the amino acid numbering used for the biochemical and structural data is that defined by Wang and Weiner (5)) on the aldehydic function followed by hydride transfer that leads to formation of a thioacylenzyme intermediate and NAD(P)H. This intermediate then undergoes a nucleophilic attack by an activated water or CoA molecule. Over the past 15 years both mechanistic and structural aspects of hydrolytic ALDHs have been studied extensively (5)(6)(7)(8)(9)(10)(11)(12)(13). In addition to local conformational reorganizations of the active site induced by ligand binding that provide the required flexibility for an efficient catalysis (14,15), one of the key aspects of the chemical mechanism of this ALDH family is the substantial conformational flexibility of the NMN moiety of the cofactor and in particular of the nicotinamide ring.…”

Mechanistic Implications of the Cysteine−Nicotinamide Adduct in Aldehyde Dehydrogenase Based on Quantum Mechanical/Molecular Mechanical Simulations

“…sequentially adjacent cysteines are located in the active site; the central member, Cys302, represents the reactive nucleophile (2). (The flanking cysteines are not highly conserved).…”

Novel Dehydrogenase Catalyzes Oxidative Hydrolysis of Carbon-Nitrogen Double Bonds for Hydrazone Degradation

“…4B) (26). A glutamate residue might function in both or either of the steps in Aldh (26). Here we showed that the E267A mutant of Hdh possessed only 0.4% of the normal level of Hdh, suggesting that Glu 267 functions as the general base.…”

“…Functional Relation between Hdh and Aldh-The deduced amino acid sequence as well as the effect of mutations in Hdh resembled those of Aldh (26,27). We therefore compared the Aldh activities of Hdh and S. cerevisiae Ald4p.…”