In the presence of
phenylalanine and molecular
oxygen, activated
phenylalanine hydroxylase catalyzes the oxidation of
tetrahydrobiopterin. The oxidation of this
tetrahydropterin cofactor also proceeds if the substrate,
phenylalanine, is replaced by its product,
tyrosine, in the initial reaction mixture. These two reactions have been defined as coupled and uncoupled, respectively, because in the former reaction 1 mol of
phenylalanine is hydroxylated for every mole of
tetrahydrobiopterin oxidized, whereas in the latter reaction there is no net hydroxylation of
tyrosine during the oxidation of the
tetrahydropterin. During the course of the coupled oxidation of
tetrahydrobiopterin, a
pterin 4a-carbinolamine intermediate can be detected by ultraviolet spectroscopy (Kaufman, S. (1976) in
Iron and
Copper Proteins (Yasunobu, K. T., Mower, H. F., and Hayaishi, O., eds) pp. 91-102, Plenum Publishing Corp., New York). Dix and Benkovic (Dix, T. A., and Benkovic, S. J. (1985) Biochemistry 24, 5839-5846) have postulated that the formation of this intermediate only occurs when the oxidation of the
tetrahydropteridine is tightly coupled to the concomitant hydroxylation of the
aromatic amino acid. However, during the
tyrosine-dependent uncoupled oxidation of
tetrahydrobiopterin by
phenylalanine hydroxylase, we have detected the formation of a spectral intermediate with ultraviolet absorbance that is essentially identical to that of the carbinolamine. Furthermore, this absorbance can be eliminated by the addition of
4a-carbinolamine dehydratase, an
enzyme which catalyzes the
dehydration of the 4a-carbinolamine. Quantitation of this intermediate suggests that there are two pathways for the
tyrosine-dependent uncoupled oxidation of
tetrahydrobiopterin by
phenylalanine hydroxylase because only about 0.3 mol of the intermediate is formed per mol of the cofactor oxidized.