Arrio-Dupont M
Eur J Biochem. 1978 Nov 15;91(2):369-78. doi: 10.1111/j.1432-1033.1978.tb12689.x.
At pH 8.3, the fluorescence spectrum of apoaspartate aminotransferase is characteristic of buried tryptophans (maximum at 330 nm and width at half-height equal to 51 nm). Its quantum yield is 1.69 times larger than for tryptophan in H2O and the mean decay time is 2.5 ns for the fluorescence emitted at wavelengths higher than 335 nm. Polarization of excitation spectrum (minimum at 305 nm for an emission at 360 nm), suggests an inter-tryptophan energy transfer. Accessibility to a quencher of fluorescence indicates that 34% of the fluorescence can be extinguished by iodide with a quenching constant of 4 M-1; as shown by solvent perturbation spectroscopy, this partial accessibility is related to two tryptophan residues accessible to solvent. At pH 5, the relative quantum yield is slightly lower than at pH 8.3 (1.65). Binding of the pyridoxal-P coenzyme diminishes the fluorescence quantum yield relative to tryptophan to 0.51 at pH 8.3 and 0.595 at pH 5; the decrease is smaller in the presence of pyridoxamine-P. Since the fluorescence of the coenzyme is very weak it is difficult to observe its emission sensitized by tryptophan, nevertheless, since the quenching is larger for pyridoxal-P that absorbs at 360 nm than for reduced pyridoxal-P that absorbs at 330 nm, it is deduced that the energy is transferred preferentially from exposed tryptophans. It is proposed that conformational changes in the vicinity of buried tryptophans are responsible for the remaining quenching. This hypothesis of conformational changes induced by the binding of the coenzyme is in agreement with the observed fluorescence emission of tyrosine. In the apoenzyme the tyrosine quantum yield is zero and the energy is entirely transferred to tryptophan. In the holoenzyme the quantum yield is low and the efficiency of transfer to tryptophan is 0.13 in pyridoxal-P form and 0.7 in pyridoxamine-P form. According to the Förster theory of long-range energy transfer, a change of transfer efficiency can be attributed to a modification either of the mutual orientation of tyrosine and tryptophan residues or of the distance between these residues: both interpretations correspond to a conformational change.
在pH 8.3时，脱辅基天冬氨酸转氨酶的荧光光谱具有埋藏色氨酸的特征（最大发射波长在330 nm，半高宽为51 nm）。其量子产率比水中色氨酸的量子产率大1.69倍，对于波长高于335 nm处发射的荧光，平均衰减时间为2.5 ns。激发光谱的偏振（在360 nm发射时，305 nm处有最小值）表明存在色氨酸间的能量转移。荧光猝灭剂的可及性表明，34%的荧光可被碘化物猝灭，猝灭常数为4 M⁻¹；如溶剂扰动光谱所示，这种部分可及性与两个可被溶剂接触的色氨酸残基有关。在pH 5时，相对量子产率略低于pH 8.时（1.65）。吡哆醛 - P辅酶的结合使相对于色氨酸的荧光量子产率在pH 8.3时降至0.51，在pH 5时降至0.595；在存在吡哆胺 - P时，下降幅度较小。由于辅酶的荧光非常弱，很难观察到其由色氨酸敏化的发射，然而，由于在360 nm处吸收的吡哆醛 - P的猝灭比在330 nm处吸收的还原型吡哆醛 - P的猝灭更大，因此推断能量优先从暴露的色氨酸转移。有人提出，埋藏色氨酸附近的构象变化是剩余猝灭的原因。这种由辅酶结合引起构象变化的假说与观察到的酪氨酸荧光发射一致。在脱辅基酶中，酪氨酸量子产率为零，能量完全转移到色氨酸。在全酶中，量子产率较低，在吡哆醛 - P形式下转移到色氨酸的效率为0.13，在吡哆胺 - P形式下为0.7。根据Förster远程能量转移理论，转移效率的变化可归因于酪氨酸和色氨酸残基相互取向的改变或这些残基之间距离的改变：这两种解释都对应于构象变化。
