Native and Phosphorylated Bovine Adrenal Tyrosine 3-Monooxygenase. Interactions with Tetrahydropterins and Substrate and Stability of the Formed 4a-Hydroxy-Tetrahydrobiopterin

Abstract

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.