Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum.
Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum.
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DOI：
10.1016/s0021-9258(19)75954-7
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发表时间：
1987-01
期刊：
影响因子：
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通讯作者：
G. Sancar;M. Jorns;G. Payne;D. Fluke;C. S. Rupert;A. Sancar
中科院分区：
文献类型：
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作者：
G. Sancar;M. Jorns;G. Payne;D. Fluke;C. S. Rupert;A. Sancar
测定了大肠杆菌DNA光裂合酶的体外绝对作用光谱。在体内，光复活截面（Φ）为2.4 × 10（4）M-1 cm-1，表明如果假设酶在体内具有与纯化至均一的酶相同的光谱特性（例如Φ 384 = 1.8 × 10（4）M-1 cm-1），则量子产率（Φ）约为1.0。纯酶（含FAD中性半醌自由基的蓝色酶）的相对作用光谱与光复活E. coli，λ max = 384 nm。然而，蓝酶的绝对作用光谱产生的光复活截面（在384 nm处的λ phi = 1.2 × 10（3））比体内值低20倍，表明体外量子产率明显较低（phi约等于0.07）。用连二亚硫酸盐还原酶导致黄素半醌的还原和伴随的量子产率的12-15倍增加。这些结果表明，酶的黄素辅因子在体内被完全还原，并且在吸收300-500 nm范围内的单个光子时，光解酶发色团（由还原的FAD加上第二发色团组成）向嘧啶二聚体提供电子，导致其逆转为两个嘧啶。还原的发色团在光化学步骤结束时再生，从而使酶能够起催化作用。
The absolute action spectrum of Escherichia coli DNA photolyase was determined in vitro. In vivo the photoreactivation cross-section (epsilon phi) is 2.4 X 10(4) M-1 cm-1 suggesting that the quantum yield (phi) is about 1.0 if one assumes that the enzyme has the same spectral properties (e.g. epsilon 384 = 1.8 X 10(4) M-1 cm-1) in vivo as those of the enzyme purified to homogeneity. The relative action spectrum of the pure enzyme (blue enzyme that contains FAD neutral semiquinone radical) agrees with the relative action spectrum for photoreactivation of E. coli, having lambda max = 384 nm. However, the absolute action spectrum of the blue enzyme yields a photoreactivation cross-section (epsilon phi = 1.2 X 10(3) at 384 nm) that is 20-fold lower than the in vivo values indicative of an apparent lower quantum yield (phi approximately equal to 0.07) in vitro. Reducing the enzyme with dithionite results in reduction of the flavin semiquinone and a concomitant 12-15-fold increase in the quantum yield. These results suggest that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300-500 nm range, the photolyase chromophore (which consists of reduced FAD plus the second chromophore) donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. The reduced chromophore is regenerated at the end of the photochemical step thus enabling the enzyme to act catalytically.+