7-Substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency Mechanism of formation via non-enzymatic transformation from 6-substituted pterins

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1’,2’-dihydroxypropy1)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki z 8 pM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine. L-Tetrahydrobiopterin is the natural cofactor of pterindependent mammalian monooxygenases such as phenylalanine-4-hydroxylase [l], tyrosine-3-hydroxylase and tryptophan-5-hydroxylase [2, 31. The last two enzymes play a key role in the biosynthesis of the neurotransmitters dopamine, serotonin, epinephrine and norepinephrine [4]. The absence of phenylalanine hydroxylase activity leads to classical phenylketonuria (PKU) [5]. Two additional forms of PKU are known to be caused by enzyme defects involved in the de novo biosynthesis of L-tetrahydrobiopterin [6, 71, one by a defect in the