Incorporation of molecular oxygen into pyrimidine cofactors by phenylalanine hydroxylase.

The 5-amino substituents of two pyrimidine cofactors of rat liver phenylalanine hydroxylase, 2,5,6-triamino-4-pyrimidinone (TP) and 5-benzylamino-2,6-diamino-4-pyrimidinone (BDP), have been shown to be cleaved quantitatively by enzyme (Bailey, S. W., and Ayling, J. E. (1980) J. Biol. Chem. 255, 7774-7781). That the pyrimidine product of this process (when carried out in the presence of 2-mercaptoethanol) is 2,6-diamino-5-hydroxy-4-pyrimidinone (divicine) is further confirmed by mass spectrometry of an isolated t-butyldimethylsilyl derivative. The origin of the oxygens in this divicine was studied with enzyme reactions containing 18O2. Corrected for the loss in the controls, the divicine generated by phenylalanine hydroxylase from TP and BDP incorporated one atom of 18O with an efficiency of 98 +/- 5% and 100 +/- 3%, respectively, even though these reactions are partially uncoupled. The position of the isotope was unambiguously assigned to the 5-hydroxyl group by the simultaneous use of [18O] TP and 18O2, the divicine from which was found to be doubly labeled. o-Methylphenylalanine stimulates a rate of cofactor oxidation at least 10-fold greater than its own rate of hydroxylation. The majority of divicine isolated from phenylalanine hydroxylase incubations with o-methyl substrate analog was labeled with oxygen from 18O2. The demonstration, with phenylalanine hydroxylase, that one atom of molecular oxygen remains attached to position 5 of pyrimidine cofactor, provides the first strong evidence for activation of oxygen by aromatic amino acid monooxygenases via covalent addition to C4a of tetrahydrobiopterin.