Quenching of the intrinsic fluorescence of liver alcohol dehydrogenase by the alkaline transition and by coenzyme binding.
The fluorescence of alcohol dehydrogenase is quenched by the acid dissociation of some group on the protein having an apparent pKa of 9.6 at 25 degrees C. The pKa of this alkaline quenching transition is unchanged by the binding of trifluoroethanol or pyrazole to the enzyme or by the selective removal of the active site of Zn2+ ion. This indicates that the ionization of a zinc-bound water molecule is not responsible for the quenching. The binding of NAD+ to the enzyme causes a drop in protein fluorescence and an apparent shift in the alkaline quenching transition to lower pH. In the ternary complex formed with NAD+ and trifluoroethanol the alkaline transition is difficult to discern between pH 6 and pH 11. In the NAD+-pyrazole ternary complex, however, a small but noticeable fluorescence transition is observed with a pKa(app) approximately 9.5. We propose that the alkaline transition centered at pH 9.6 is not shifted to lower pH upon binding NAD+. Instead, the amplitude of the alkaline quenching effect is decreased to the point that it is difficult to detect when NAD+ is bound. We present a model that describes the dependence of the fluorescence of the protein on pH and NAD+ concentration in terms of two independently operating, dynamic quenching mechanisms. Our data and model cast serious doubt on the identification, made previously in the literature, between the alkaline quenching pKa and the pKa of the group whose ionization is coupled to NAD+ binding.(ABSTRACT TRUNCATED AT 250 WORDS)
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D009243:NAD / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D009243:NAD; D000426:Alcohol Dehydrogenase