Abstract
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.
Highlights
Dihydropteridine reductase from sheep liver was purified through the calcium phosphate gel step (24). 4a-Carbinolamine dehydratase was purified by the method of Huang et al (21)
Phenylalanine hydroxylase was purified by the hybrid procedures as described previously (25)
Optimal Conditions for the Formation of the Carbinolamine-Untilnow, we have reported thatthe increase in absorbance at 246 nm, due to the formation of the carbinolamine during the tyrosine-dependent oxidation ofBH4 by phenylalanine hydroxylase, is significantly lower than the amount observed during the fully coupled reaction without trying to explain this difference
Summary
ANDDISCUSSION (from Leuconostoc mesenteroides),~-3,4-dihydrophenylalanine2,,4,5trihydroxyphenylalanine,tyrosinase (from mushroom), and NADH were purchased from Sigma. Biopterin is formed (17, 18).This oxidation product of the reducedpteridine is released from the enzyme and forms qBHz by losing a molecule of water (Fig. 1). this dehydration occurs rapidly under physiological conditions (17), an enzyme, 4a-carbinolamine dehydratase, catalyzes the reaction (19-22) It is qBHz which is the substrate for the NADHdependent dihydropteridine reductase, the enzyme responsible for maintaining biopterin in the fully reduced state (i.e. as BH,) (8, 9). The extrapolation was performed for the fully coupled reaction, but in the absence of a regenerating system for qBH2 After this extinction coefficient was determined, the spectrum of the carbinolamine was obtained from the deconvolution of spectra which included only the carbinolamine and BH4 (i.e. at early times prior to formation of significant amounts of qBH2). An Intermediate in the UncoupOlexdidation of Tetrahydrobiopterin tO.*O a ' I I i
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