Benci S, Vaccari S, Mozzarelli A, Cook P F
Institute of Physical Sciences, University of Parma, Italy.
Biochim Biophys Acta. 1999 Jan 11;1429(2):317-30. doi: 10.1016/s0167-4838(98)00229-5.
Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at lamda > 470 nm, indicates the presence of two components characterized by lifetimes (tau) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400-560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved; (v) the increased emission at 490 nm, caused by acetate binding, is likely due to the stabilization of the ketoenamine tautomer induced by an increase in polarity of the active site microenvironment and/or a decrease in proton dissociation in the excited state; (vi) excitation at 330 nm, where the enolimine tautomer absorbs, leads to emission decays typical of the ketoenamine.
已对磷酸吡哆醛（PLP）依赖性酶O - 乙酰丝氨酸巯基酶（OASS）的内部醛亚胺、游离PLP以及PLP - L - 缬氨酸席夫碱的静态和时间分辨荧光进行了测量，以深入了解与OASS结合的PLP的光物理性质。在330 nm处激发时，游离辅酶在415 nm处呈现一个谱带，而PLP - 缬氨酸和OASS（在其吸收最大值处激发时也是如此）呈现出一种结构化发射，其峰值在420 nm，在490和530 nm处有肩部。420和490 nm处的发射带分别归因于内部醛亚胺的烯醇亚胺和酮烯胺互变异构体，而530 nm处的发射可能源于激发态质子解离时形成的偶极物种。在412 nm（415 nm）处激发并在λ>470 nm处收集的OASS（PLP - 缬氨酸）的时间分辨荧光表明存在两个组分，其特征寿命（τ）分别为0.6（0.08）和3.8（1.55）ns，具有相等的分数强度（f）。在乙酸盐存在下，慢组分在OASS发射中占主导，f为0.98。在350 nm处激发作为发射波长（400 - 560 nm）的函数显示至少有三个组分。慢组分的f从400 nm增加到440 nm，然后降低，而中间和快组分的f表现相反。结果表明：（i）快组分与530 nm处的发射相关；（ii）慢组分与420 nm处的发射相关；（iii）在发射光谱的蓝侧存在一个寿命非常短的快速加和组分；（iv）中间组分是由重叠贡献导致的，包括无法分辨的490 nm处谱带的发射；（v）乙酸盐结合导致的490 nm处发射增加可能是由于活性位点微环境极性增加和/或激发态质子解离减少诱导的酮烯胺互变异构体的稳定；（vi）在330 nm处激发，此时烯醇亚胺互变异构体吸收，导致酮烯胺典型的发射衰减。
