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.
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.
Motion
R L
RL
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Buckley
P D
PD
Bennett
A F
AF
Blackwell
L F
LF
D000079:Acetaldehyde / Q000378:metabolism; D000089:Acetic Anhydrides / Q000378:metabolism; D000215:Acylation; D000444:Aldehyde Dehydrogenase / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D001665:Binding Sites; D006868:Hydrolysis; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism*; D010084:Oxidation-Reduction
D000089:Acetic Anhydrides; D000447:Aldehydes; D046911:Macromolecular Substances; D009596:Nitrophenols; D009243:NAD; C031800:acetic anhydride; C008642:4-nitrophenyl acetate; D000444:Aldehyde Dehydrogenase; D000079:Acetaldehyde