Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases.
The horse EE and human beta 1 beta 1 alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp-15 and Trp-314). Tyr-286, which had been proposed to quench the fluorescence of Trp-314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in beta 1 beta 1. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of beta 1 beta 1 is not substantially quenched at alkaline pH. Tyr-286 had also been implicated in the quenching of Trp-314 upon formation of the EE-NAD(+)-trifluoroethanol ternary complex. However, beta 1 beta 1 exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr-286 is not involved in ternary complex quenching.
Ehrig
T
T
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46202-5122.
Muhoberac
B B
BB
Hurley
T D
TD
Bosron
W F
WF
D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D000818:Animals; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry* / Q000502:physiology
D007527:Isoenzymes; D014364:Tryptophan; D000426:Alcohol Dehydrogenase