Hatakeyama K, Hoshiga M, Suzuki S, Kagamiyama H
Department of Medical Chemistry, Osaka Medical College, Japan.
Anal Biochem. 1993 Feb 15;209(1):1-5. doi: 10.1006/abio.1993.1074.
An enzymatic method for preparing 6R-[U-14C]-tetrahydrobiopterin from [U-14C]GTP is presented. This method utilizes purified preparations of three biosynthetic enzymes for 6R-tetrahydrobiopterin, i.e., Escherichia coli GTP cyclohydrolase I, rat 6-pyruvoyl-tetrahydropterin synthase, and rat sepiapterin reductase. Based on the catalytic properties of these enzymes, the reaction conditions were optimized to complete the entire conversion reaction from GTP to 6R-tetrahydrobiopterin in a single reaction solution without the need to isolate unstable intermediates after each enzymatic reaction. The reaction conditions thereby established yielded [U-14C]biopterin in an amount equivalent to 75%, on a molar basis, of the initial amount of [U-14C]GTP. The product was subsequently isolated by high-performance liquid chromatography. The method produced labeled 6R-tetrahydrobiopterin with a specific activity of 450 Ci/mol and an overall yield of more than 60%.
本文介绍了一种从[U-14C]GTP制备6R-[U-14C]-四氢生物蝶呤的酶法。该方法利用了三种用于合成6R-四氢生物蝶呤的生物合成酶的纯化制剂，即大肠杆菌GTP环化水解酶I、大鼠6-丙酮酸四氢蝶呤合酶和大鼠蝶啶还原酶。基于这些酶的催化特性，对反应条件进行了优化，以便在单一反应溶液中完成从GTP到6R-四氢生物蝶呤的整个转化反应，而无需在每次酶促反应后分离不稳定的中间体。由此确定的反应条件产生的[U-14C]生物蝶呤的量，以摩尔计，相当于初始[U-14C]GTP量的75%。随后通过高效液相色谱法分离产物。该方法产生的标记6R-四氢生物蝶呤的比活度为450 Ci/mol，总产率超过60%。
