Haavik et al.: Oxidation of tetrahydropterins by tyrosine hydroxylase 11 Pteridines Vo1.1, 1989, pp.11-16 Native and Phosphorylated Bovine Adrenal Tyrosine 3-Monooxygenase. Interactions with Tetrahydropterins and Substrate and Stability of the Formed 4a-Hydroxy- Tetrahydrobiopterin By Jan Haavik*, Kristoffer K. Andersson, Torgeir Flatmark Department of Biochemistry, University of Bergen , Arstadveinen, 19, N-5009 Bergen, Norway Leif Petersson Department of Biophysics, University of Stockholm, S-1 0691 Stockholm, Sweden (Received May 1988) Summary The catalytic activity of tyrosine 3-monooxygenase (tyrosine hydroxylase) is dependent on a tetrahydropterin cofactor and ezyme-bound iron . The oxidation of tetrahydrobiopterin by bovine adrenal tyrosine hydroxylase was studied by high performance liquid chromatography (HPLC). The first pterin product detected during catalytic turnover, 4a-hydroxy-tetrahydrobiopterin, was isolated by HPLC and the pseudo first-order rate constant of its dehydration to quinonoid dihydrobiopterin was estimated. The tl /2 was found to be 45 and 72 s in 100 and 5 mmol /L Tris-HCl, respectively, at pH 7.5 and 23 °C. The rate of the 4a-hydroxy-tetrahydropterin formation was found to increase on phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. The phosphorylation did not significantly change the electron paramagnetic resonance (EPR) spectrum of tyrosine hydroxylase. However, in the presence of substrate or cofactor and dithiothreitol, the EPR spectral properties were strikingly different from those observed prior to phosphorylation. Our findings are in accordance with a mechanism in which the covalently bound phosphate activates the enzyme by facilitating changes in the redox-state of the enzyme-bound iron. Introduction The most well-defined biological role of tetrahydrop- terins in mammals is their function as electron donors in the monooxygenase reactions catalyzed by the three aromatic amino acid hydroxylases , i. e. phenylalanine , tyrosine and tryptophan hydroxylase (for reviews, see refs. 1 - 3). These enzymes show a high degree of * Author to whom correspondence should be addressed Enzymes: Tyrosine 3-monooxygenase, tyrosine hydroxyla se (EC 1.14.16.2); Phenylalanine 4-monooxygenase, phenylalanine hy- droxylase (EC 1.14.16.1). Abbreviations: BH 4 , 6(R)-tetrahydrobiopterin; 4a-OH-BH4, 4a- hydroxy-tetrahydrobiopterin; qBH 2 , quinonoid dihydrobiop- terin ; 7,8-BH 2 , 7,8-dihydrobiopterin; 6-MPH 4 , 6-methyltetra- hydropterin ; q6-MPH 2 , quinonoid 6-methyldihydropterin ; DOPA, 3,4-dihydroxy-phenylalanine; DTT, dithiothreitol ; E PR, electron paramagnetic resonance ; HPLC, high-perform- ance liquid chromatography. Pteridines / Vo1. 1 . 1989 Copyright (0 1989 \\ "a lt er de Gruytcr " Berlin " York sequence homology and are postulated to have similar reaction mechanisms (1 -4). During catalytic turn- over, tetrahydropterins are oxidized to dihydropterins in the tyrosine hydroxylase reaction (1 - 3, 5), e. g. k, L- Tyrosine + BH4 + O2 -t DOPA + 4a-OH-BH4 (1 ) k, 4a-OH-BH4 -t qBH 2 + H 2 O (2) k, qBH2 -t 7,8-BH 2 (3) The formed dihydrobiopterins (qBH2 and 7,8-BH2) can in turn be regenerated to tetrahydrobiopterin (BH4) by dihydropteridine reductase and dihydrofol- ate reductase , respectively (2). Although the overall stoichiometry of the hydroxylation reaction (equation 1) is known , the precise function of the tetrahydrop- Unauthenticated Download Date | 7/28/18 8:32 PM