Multiphoton Manipulations of Enzymatic Photoactivity in Aspartate Aminotransferase
Multiphoton Manipulations of Enzymatic Photoactivity in Aspartate Aminotransferase
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DOI：
10.1021/jp112400g
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发表时间：
2011-04-21
影响因子：
3.3
通讯作者：
Larsen, Delmar S.
中科院分区：
文献类型：
--
作者：
Hill, Melissa P.;Freer, Lucy H.;Larsen, Delmar S.
天冬氨酸氨基转移酶（AAT）利用发色团吡哆醛S '-磷酸（PLP）辅因子促进氨基酸的转氨作用。最近，我们证明，在暴露于蓝光时，PLP形成反应性三重态，当与PLP依赖性酶结合时，该反应性三重态快速（以微秒计）产生高能喹喔啉中间体[J. Am. 2010，132（47），16953-16961]。这增加了AAT的净催化活性（k（cat）），因为醌的形成是通过热活化的酶促途径的部分速率限制。所观察到的光增强的幅度最初与泵注量成线性比例;然而，当超过临界阈值时，光活性饱和，甚至在更大的激发注量下被抑制。与这种抑制行为相关联的光动力学机制的特征在于使用超快多脉冲泵浦-转储-探测和泵浦-再泵浦-探测瞬态吸收技术结合互补双色，稳态激发测定。通过对瞬态超快数据和稳态分析数据的多态动力学建模，将AAT中光活性的非单调入射功率依赖性分解为激发单重态的高强度倾倒和激发三重态的再泵浦的贡献，并通过更高三重态电子流形中的快速系间穿越诱导基态的再布居。
The aspartate aminotransferase (AAT) enzyme utilizes the chromophoric pyridoxal S'-phosphate (PLP) cofactor to facilitate the transamination of amino acids. Recently, we demonstrated that, upon exposure to blue light, PLP forms a reactive triplet state that rapidly (in microseconds) generates the high-energy quinonoid intermediate when bound to PLP-dependent enzymes [J. Am. Chem. Soc. 2010, 132 (47), 16953-16961]. This increases the net catalytic activity (k(cat)) of AAT, since formation of the quinonoid is partially rate limiting via the thermally activated enzymatic pathway. The magnitude of observed photoenhancement initially scales linearly with pump fluence; however when a critical threshold is exceeded, the photoactivity saturates and is even suppressed at greater excitation fluences. The photodynamic mechanisms associated with this suppression behavior are characterized with the use of ultrafast multipulse pump-dump-probe and pump-repump-probe transient absorption techniques in combination with complementary two-color, steady-state excitation assays. Via multistate kinetic modeling of the transient ultrafast data and the steady-state assay data, the nonmonotonic incident power dependence of the photoactivty in AAT is decomposed into contributions from high-intensity dumping of the excited singlet state and repumping of the excited triplet state with induces the repopulation of the ground state via rapid intersystem crossing in the higher-lying triplet electronic manifold.