Hamm-Alvarez S, Sancar A, Rajagopalan K V
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1990 Oct 25;265(30):18656-62.
Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.
大肠杆菌DNA光解酶催化光驱动（300 - 500纳米）修复紫外线（200 - 300纳米）照射下DNA中相邻嘧啶碱基之间形成的嘧啶二聚体。光驱动修复过程由两种与酶结合的辅因子FADH2和5,10 - 亚甲基四氢叶酸促进。在这一系列实验中，对叶酸的功能进行了更详细的表征。对光解酶与5,10 - 亚甲基四氢叶酸的单谷氨酸和多谷氨酸形式的相对结合亲和力的研究表明，该酶对叶酸的天然存在的多谷氨酸形式具有更高的亲和力，并且外源添加的单谷氨酸衍生物与蛋白质的结合较松散。多次周转实验表明，即使在酶进行10次周转后，叶酸仍与光解酶结合。在多次周转条件下以及微摩尔浓度的酶存在或不存在游离叶酸的情况下，对含有化学计量叶酸的光解酶的修复速率进行检查，也证明叶酸起催化作用。在低浓度光解酶下观察到的外源叶酸对周转的刺激作用被证明是由于光解酶对用于重组程序的单谷氨酸衍生物的亲和力较低。这些结果表明，大肠杆菌DNA光解酶的叶酸是一种真正的辅因子，并且在酶的多次周转过程中不会分解或解离。
