Waldman A D, Clarke A R, Wigley D B, Hart K W, Chia W N, Barstow D, Atkinson T, Munro I, Holbrook J J
Biochim Biophys Acta. 1987 May 27;913(1):66-71. doi: 10.1016/0167-4838(87)90233-0.
Site-directed mutagenesis has been used to generate two mutant Bacillus stearothermophilus lactate dehydrogenases: in one, Trp-150 has been replaced with a tyrosine residue and, in the other, both Trp-150 and -80 are replaced with tyrosines. Both enzymes are fully catalytically active and their affinities for substrates and coenzymes, and thermal stabilities are very similar to those of the native enzyme. Time-resolved fluorescence measurements using a synchrotron source have shown that all three tryptophans in the native enzyme fluoresce. By comparing the mutant and native enzymes it was possible, for the first time, to assign, unambiguously, lifetimes to the individual tryptophans: Trp-203 (7.4 ns), Trp-80 (2.35 ns) and Trp-150 (less than 0.3 ns). Trp-203 is responsible for 75-80% of the steady-state fluorescence emission, Trp-80 for 20%, and Trp-150 for less than 2%.
一种是将色氨酸-150替换为酪氨酸残基，另一种是将色氨酸-150和-80都替换为酪氨酸。这两种酶都具有完全的催化活性，它们对底物和辅酶的亲和力以及热稳定性与天然酶非常相似。使用同步辐射源进行的时间分辨荧光测量表明，天然酶中的所有三个色氨酸都会发出荧光。通过比较突变酶和天然酶，首次能够明确地为各个色氨酸确定寿命：色氨酸-203（7.4纳秒）、色氨酸-80（2.35纳秒）和色氨酸-150（小于0.3纳秒）。色氨酸-203占稳态荧光发射的75 - 80%，色氨酸-80占20%，色氨酸-150占不到2%。
