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A transient release of protons with an amplitude corresponding to one proton per active site has been observed for the oxidation of propionaldehyde, acetaldehyde, and benzaldehyde by sheep liver cytoplasmic aldehyde dehydrogenase at pH 7.6 with phenol red as indicator. At saturating substrate levels, the rate constants for the proton burst are in each case the same, and for acetaldehyde and propionaldehyde show the same dependence on the concentrations of the substrates, as the rate constants for the transient production of NADH reported previously [MacGibbon, A.K.H., Blackwell, L.F., & Buckley, P.D. (1977) Biochem. J. 167, 469-477]. Although, with propionaldehyde as a substrate, a full proton burst is also observed at pH 6.0, no proton burst is observed at pH 9.0. For 4-nitrobenzaldehyde, there is no burst in NADH production, but a burst in proton release is observed, showing that proton release precedes hydride transfer. No protons were released during the binding of the substrate analogues acetone and chloral hydrate nor on reaction of the enzyme with the inhibitor tetraethylthiuram disulfide (disulfiram). A model is proposed in which the rate-limiting step in the pre-steady-state phase of the reaction is a conformational change which occurs after the binding of aldehydes to the enzyme. As a result of the conformational change, the environment of a functional group on the enzyme, which initially has a pKa of about 8.5, is perturbed to give a final pKa value for the group of less than 5. Computer simulations were used to show that the model accurately reproduces all of the experimental data. The lack of observation of a second transient proton release, as required by the overall stoichiometry, argues that its release occurs in a slow step prior to NADH dissociation.

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1. Cytoplasmic aldehyde dehydrogenase was shown to be free of contamination by the mitochondrial enzyme by isoelectric focusing.2. Both enzymes showed inultiple banding in activity stains. The cytoplasmic enzyme gave two very close bands pI = 5.22 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pI 3. Disulfiram in a fourfold excess reduced the activity of the cytoplasmic enzyme to 9 2, of the initial valuc.The residual activity represents the activity of the disulfiram-modified enzyme and is not due to mitochondrial contamination. This casts doubt on the role of an essential thiol group. 4. The mitochondrial enzyme shows a low amplitude (22'11() burst in the production of 4-nitrophenoxide ion during the hydrolysis of 4-nitrophenyl acetate at pH 7.6. The burst rate constant was 7.3 & 1 s and the steady-state rate constant was 0.2 s-I , values similar to those previously reported for the cytoplasinic enzyme.5. The mitochondrial enzyme shows a burst in the release of protons during thc oxidation of propionaldehyde at pH 7.6. The burst rate constant was 6 sC1 and.the amplitude was equal to half the formal enzyme concentration.

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