Enzymatic synthesis of 6R-[U-14C]tetrahydrobiopterin from [U-14C]GTP.
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%.
Hatakeyama
K
K
Department of Medical Chemistry, Osaka Medical College, Japan.
Hoshiga
M
M
Suzuki
S
S
Kagamiyama
H
H
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000201:enzymology; D006136:GTP Cyclohydrolase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D010651:Phenylalanine Hydroxylase / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases; D051381:Rats; D013997:Time Factors
D002250:Carbon Radioisotopes; D001708:Biopterins; D006160:Guanosine Triphosphate; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; D010651:Phenylalanine Hydroxylase; D006136:GTP Cyclohydrolase; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; C003402:sapropterin