Proton release during the pre-steady-state oxidation of aldehydes by aldehyde dehydrogenase. Evidence for a...

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.