The mechanism of quenching of liver alcohol dehydrogenase fluorescence due to ternary complex formation.
Difference fluorescence emission spectra, reciprocal Stern-Volmer plots, and variable excitation wave-lengths have been used to evaluate the selective quenching of the two tryptophan residues/subunit of liver alcohol dehydrogenase. Trp-15, at the surface of the enzyme, is quenched by KI consistent with a collisional mechanism, and has a blue-shifted excitation and red-shifted emission spectrum when compared with the spectral properties of TRP-314, which is in a hydrophobic milieu at the subunit interface of the dimeric enzyme. With excitation at 295 nm, Trp-314 is 80% quenched by formation of a ternary enzyme.NAD+.trifluoroethanol complex, and the quenching is essentially additive to that caused by KI. Alkaline pH also results in selective quenching of Trp-314. These results, and considerations of the three-dimensional structure of the enzyme, indicate that the quenching of protein fluorescence of liver alcohol dehydrogenase by either ternary complex formation or alkaline pH is due to resonance energy transfer to tyrosinate. Likely candidates as energy acceptors are the Tyr-286 residues are within transfer distance for each Trp-314 residue, as well as being at the surface of the enzyme and 30 A from the active center zinc atom. Alkaline pH directly ionizes this tyrosine residue, while ternary complex formation causes a conformational change resulting in its ionization.
D000429:Alcohol Oxidoreductases; D000818:Animals; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D046911:Macromolecular Substances; D014364:Tryptophan; D000429:Alcohol Oxidoreductases