Rapid-scanning spectral evidence for catalytically nonequivalent but interconvertible forms of equine liver alcohol dehydrogenase.
These rapid-scanning stopped-flow kinetic studies of the equine liver alcohol dehydrogenase-catalyzed reduction of p-nitrobenzaldehyde by NADH and (4R)-4-deuterio NADH (NADD) under single turnover conditions establish : (1) The reaction is biphasic using NADD as coenzyme, k1 approximately 200 sec-1, k2 = 0.5 sec-1 and the amplitude ratio (A1)/(A1 + A2) approximately equal to 0.5. (2) Each phase of the reaction involves the oxidation of enzyme-bound reduced coenzyme. (3) The recycling of sites in the presence of 20 mM pyrazole is negligible. (4) The rates of E(NAD-pyrazole) complex formation at all sites and oxidation of reduced coenzyme in the slow phase are limited by the same process. We conclude that the biphasicity arises from nonequivalent but interconvertible forms of the enzyme, and that interconversion of the two forms is mediated by ligand-dependent protomer-protomer interactions.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001547:Benzaldehydes / Q000378:metabolism; D001665:Binding Sites; D006736:Horses / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008823:Micelles; D009243:NAD / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D001547:Benzaldehydes; D008823:Micelles; D009243:NAD; C029915:4-nitrobenzaldehyde; D000429:Alcohol Oxidoreductases