Jullien M, Baudet S, Rodier F, Le Bras G
Laboratoire de Biologie Physico-Chimique, Université Paris-Sud, Orsay, France.
Biochimie. 1988 Dec;70(12):1807-14. doi: 10.1016/0300-9084(88)90042-9.
The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.
已通过时间分辨荧光光谱法研究了来自大肠杆菌的苏氨酸敏感型天冬氨酸激酶I-高丝氨酸脱氢酶I的变构转变。荧光衰减可分解为2种不同类型的色氨酸发射体：一种快速成分，寿命约为1.5纳秒；以及一种慢速成分，寿命约为4.5纳秒。慢速成分的荧光特性会因变构转变而改变。在由苏氨酸稳定的酶的T型中，慢速成分的寿命更长，光谱发生红移；其被丙烯酰胺猝灭的可及性略有提高，而荧光各向异性没有任何降低。这些结果表明慢速成分环境的极性发生了变化。与变构转变相关的四级结构变化可能涉及结构域的整体移动，而不会在纳秒时间尺度上导致任何局部移动。我们认为慢速成分对应于埋藏的激酶结构域中独特的色氨酸。
