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

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Abstract
Acylation of the aldehyde dehyrogenase .cntdot. 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 .cntdot. 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 .cntdot. NADH intermediate despite the rapid hydrolysis of this *enzyme .cntdot. acyl .cntdot. NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman and Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase .cntdot. 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 and 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.