Laws W R, Shore J D
J Biol Chem. 1978 Dec 10;253(23):8593-7.
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.
利用差示荧光发射光谱、倒数斯特恩-沃尔默图和可变激发波长来评估肝醇脱氢酶两个色氨酸残基/亚基的选择性猝灭。位于酶表面的Trp-15被KI猝灭，这与碰撞机制一致，并且与处于二聚体酶亚基界面疏水环境中的TRP-314的光谱特性相比，其激发光谱发生蓝移，发射光谱发生红移。在295nm激发下，通过形成三元酶.NAD⁺.三氟乙醇复合物，Trp-314有80%被猝灭，并且这种猝灭基本上是KI所引起猝灭的加和。碱性pH也会导致Trp-314的选择性猝灭。这些结果以及对该酶三维结构的考虑表明，通过形成三元复合物或碱性pH对肝醇脱氢酶蛋白质荧光的猝灭是由于共振能量转移到酪氨酸根离子。可能作为能量受体的候选者是Tyr-286残基，其与每个Trp-314残基处于转移距离之内，并且位于酶表面且距离活性中心锌原子30埃。碱性pH直接使该酪氨酸残基电离，而形成三元复合物会导致构象变化从而使其电离。
