Evidence that the cytoplasmic aldehyde dehydrogenase-catalysed oxidation of aldehydes involves a different active-site group from that which catalyses the hydrolysis of 4-nitrophenyl acetate

Abstract

Acylation of the aldehyde dehydrogenase.NADH complex by acetic anhydride leads to the production of acetaldehyde and NAD+. By monitoring changes in nucleotide fluorescence, the rate constant for acylation of the active site of the *enzyme.NADH complex was found to be 11 +/- 3 s-1. The rate of acylation by acetic anhydride at the group that binds aldehydes on the oxidative pathway is clearly rapid enough to maintain significant steady-state concentrations of the required active-site-acylated *enzyme.NADH intermediate despite the rapid hydrolysis of this *enzyme.acyl.NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman & Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase.NADH complex by 4-nitrophenyl acetate also occurs rapidly with a rate constant of 10.9 +/- 0.6 s-1, even under the most extreme trapping conditions only very small amounts of acetaldehyde are detected [Loomes & Kitson (1986) Biochem. J. 235, 617-619]. Furthermore enzyme-catalysed hydrolysis of 4-nitrophenyl acetate is limited by the rate of deacylation of a group on the enzyme (0.4 s-1), which is an order of magnitude less than deacylation of the group at the active site (5-10 s-1). It is concluded that the enzyme-catalysed 4-nitrophenyl ester hydrolysis involves a group on the enzyme that is different from the active-site group that binds aldehydes on the normal oxidative pathway.

Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase

“…There has long been disagreement as to whether ester and aldehyde substrates interact with aldehyde dehydrogenase at the same active site or not (Blackwell et al, 1983;Duncan, 1985;Loomes & Kitson, 1986;Motion et al, 1988 Kitson, 1989a,b) suggest that the first of these three possibilities is unlikely: (1) PNP dimethylcarbamate is a competitive inhibitor of the dehydrogenation of D-glyceraldehyde or propionaldehyde; (2) the presence of propionaldehyde or chloral hydrate (a competitive inhibitor of the dehydrogenase activity) slows the reaction of PNP dimethylcarbamate with the enzyme; (3) premodification of aldehyde dehydrogenase by disulfiram largely abolishes both the dehydrogenase activity of the enzyme and its action on PNP dimethylcarbamate; (4) modification of aldehyde dehydrogenase by PNP dimethylcarbamate profoundly affects the fluorescence of enzyme-bound NADH and the rate of dissociation of NADH; these results are consistent with the site of modification being the dehydrogenase active site; (5) high propionaldehyde concentration affects the fluorescence of enzyme-bound NADH, but not when the enzyme is pre-modified by PNP dimethylcarbamate, suggesting that the aldehyde substrate cannot bind to the modified enzyme. Clearly, all these points are most simply explained if the esterase and dehydrogenase reactions are mediated by the same catalytic nucleophile.…”