title: Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes.
abstract: Binding of NAD+ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependent and is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD+ complex. Deprotonation during binding of NAD+ and inhibitors that trap the enzyme-NAD+ complex was examined by transient kinetics with pH indicators, and formation of complexes was monitored by absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic proton release, whereas reaction with caprate showed proton release followed by proton uptake. Proton release (200-550 s(-1)) is a common step that precedes binding of all inhibitors. At all pH values studied, the rate constants for proton release or uptake matched those for formation of ternary complexes, and the most significant quenching of protein fluorescence (or perturbation of adenine absorbance at 280 nm) was observed for enzyme species involved in deprotonation steps. Kinetic simulations of the combined transient data for the multiple signals indicate that all inhibitors bind faster and tighter to the unprotonated enzyme-NAD+ complex, which has a pK of about 7.3. The results suggest that rate-limiting deprotonation of the enzyme-NAD+ complex is coupled to the conformational change and controls the formation of ternary complexes.
authors.affiliation: Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: Elena G
authors.initials: EG
authors.lastname: Kovaleva
authors.forename: Bryce V
authors.initials: BV
authors.lastname: Plapp
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D011522:Protons
chemical_list: D004791:Enzyme Inhibitors; D011522:Protons; D009243:NAD; D000426:Alcohol Dehydrogenase