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

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