IDENTIFICATION OF THE 2ND CHROMOPHORE OF ESCHERICHIA-COLI AND YEAST DNA PHOTOLYASES AS 5,10-METHENYLTETRAHYDROFOLATE
IDENTIFICATION OF THE 2ND CHROMOPHORE OF ESCHERICHIA-COLI AND YEAST DNA PHOTOLYASES AS 5,10-METHENYLTETRAHYDROFOLATE
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DOI：
10.1073/pnas.85.7.2046
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发表时间：
1988-04-01
影响因子：
11.1
通讯作者：
SANCAR, A
中科院分区：
文献类型：
--
作者：
JOHNSON, JL;HAMMALVAREZ, S;SANCAR, A
用盐酸胍或酸化至pH 2使来自大肠杆菌的DNA光裂合酶（脱氧核糖嘧啶光裂合酶，EC 4.1.99.3）变性，除了FAD之外，还释放出具有还原蝶呤的光谱和色谱特性的发色团。在酸化之前用碘处理酶将发色团转化为稳定的氧化衍生物，通过HPLC将其解析为具有相同光谱性质的四种物质。从酵母酶中获得相同分布的相同物种。从碘氧化酶中分离的物质通过用碱性高锰酸盐转化为蝶呤-6-羧酸而被证明是蝶呤，并且发现在酸水解时释放谷氨酸。通过吸收和荧光光谱以及通过脱乙酰基和转化为叶酸来证明在分离的氧化发色团中存在10-甲酰叶酸。聚谷氨酸盐的分布分析表明，通过HPLC鉴定的四种物质对应于10-甲酰叶酸的三-、四-、五-和六-谷氨酸盐衍生物。结果与γ一致。三谷氨酸盐衍生物中的键与通过α-谷氨酸盐连接的另外的谷氨酸盐连接前一个残基的羧基。用大鼠血浆水解酶处理产生10-甲酰叶酸的单谷氨酸衍生物。通过在低pH下进行释放和分离以保护5，10-亚甲基桥并保留还原的吡嗪环结构，将叶酸辅因子的天然酶结合形式鉴定为5，10-亚甲基四氢亚叶酸聚谷氨酸盐。
Denaturation of DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) from Escherichia coli with guanidine hydrochloride or acidification to pH 2 released, in addition to FAD, a chromophore with the spectral and chromatographic properties of a reduced pterin. Treatment of the enzyme with iodine prior to acidification converted the chromophore to a stable, oxidized derivative, which was resolved by HPLC into four species with identical spectral properties. The same species, in the same distribution, were obtained from the yeast enzyme. The material isolated from the iodine-oxidized enzyme was shown to be a pterin by conversion to pterin-6-carboxylic acid with alkaline permanganate and was found to release glutamate upon acid hydrolysis. The presence of 10-formylfolate in the isolated, oxidized chromophore was demonstrated by absorption and fluorescence spectroscopy and by deformylation and conversion to folic acid. Analysis of the distribution of polyglutamates revealed that the four species identified by HPLC corresponded to the tri-, tetra-, penta-, and hexaglutamate derivatives of 10-formylfolate. The results were consistent with .gamma. linkages in the triglutamate derivative with additional glutamates linked via the .alpha.-carboxyl group of the preceding residue. Treatment with rat plasma hydrolase produced the monoglutamate derivative of 10-formylfolate. The native, enzyme-bound form of the folate cofactor was identified as 5,10-methenyltetrahydrofolylpolyglutamate by effecting release and isolation at low pH to protect the 5,10-methenyl bridge and preserve the reduced pyrazine ring structure.