ACTIVE-SITE OF DNA PHOTOLYASE - TRYPTOPHAN-306 IS THE INTRINSIC HYDROGEN-ATOM DONOR ESSENTIAL FOR FLAVIN RADICAL PHOTOREDUCTION AND DNA-REPAIR INVITRO
ACTIVE-SITE OF DNA PHOTOLYASE - TRYPTOPHAN-306 IS THE INTRINSIC HYDROGEN-ATOM DONOR ESSENTIAL FOR FLAVIN RADICAL PHOTOREDUCTION AND DNA-REPAIR INVITRO
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DOI：
10.1021/bi00239a034
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发表时间：
1991-06-25
期刊：
影响因子：
2.9
通讯作者：
SANCAR, A
中科院分区：
文献类型：
--
作者：
LI, YF;HEELIS, PF;SANCAR, A
DNA 光解酶通过光诱导电子转移修复 DNA 中的环丁二嘧啶 (PyrPyr)。从大肠杆菌中分离出来的酶含有亚甲基四氢叶酸 (MTHF)（充当光天线）和 FADH2（氧化还原活性辅助因子）。在纯化过程中，FADH2 被氧化成蓝色中性自由基形式 FADH，其活性大大降低。先前的纳秒闪光光解研究 [Heelis, P. F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] 表明 FADH 的激发。直接通过吸收光子或间接通过从 MTHF 激发单线态转移电子能量产生 FADH。四重奏从附近的色氨酸中提取了一个氢原子，以从酶中产生具有催化能力的 FADH2。使用定点诱变，我们将所有 15 个光裂合酶色氨酸残基分别替换为苯丙氨酸，以鉴定负责光还原的内部氢原子供体。我们发现W306F突变消除了FADH的光还原作用。不影响 FADH 的激发态特性。或酶的底物结合（K(A) 约为 10(9) M-1）。在反应混合物中不存在还原剂的情况下，突变酶的特异性常数 (k(cat)/K(m)) 大约为 0，表明 FADH 发生光还原。是体外光裂解酶光修复的重要步骤。 FADH 的化学还原。突变酶的特异性常数恢复到野生型水平。
DNA photolyases repair cyclobutadipyrimidines (PyrPyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P. F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (K(A) approximately 10(9) M-1) of the enzyme. The specificity constant (k(cat)/K(m)) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.