Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human β1β1 and horse EE alcohol dehydrogenases

FEBS Letters

Ehrig, Torsten²  Bosron, William F.²  Muhoberac, Barry B.¹  Hurley, Thomas D.² 

[1] Department of Chemistry, Purdue University School of Science, Indiana University-Purdue University at Indianapolis, Indianapolis, IN 46205, USA;Departments of Biochemistry and Molecular Biology, and Medicine, Indiana University School of Medicine, Indianapolis, IN 46202-5122, USA

关键词: Alcohol dehydrogenase;    Isoenzyme;    Fluorescence spectroscopy;    Tryptophan quenching;    ADH;    alcohol dehydrogenase;    DTT;    dithiothreitol;    HEPES;    N-[2-hydroxyethylpiperazine-N′-[2-ethanesulfonic acid];    MES;    2[N-morpholino]ethanesulfonic acid;    ACES;    N-[2-acetamidol-2-aminoethanesulfonic acid;    EDTA;    ethylendiaminitetraacetic acid;   

DOI  :  10.1016/0014-5793(92)80864-D

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The horse EE and human β₁β₁ 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 β₁β₁. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of β₁β₁ 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, β₁β₁ 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.

【 授权许可】

Unknown