Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: Large-scale purification and physicochemical properties

Abstract

A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) ( l-tyrosine,tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit M r of 60 000 anda specific activity of 425 nmol · min −1 · mg −1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 ± 0.09 mol iron, 0.13 mol zinc and 0.62 ± 0.04 mol phosphate per mol subunit of M r = 60 000. A broad light absorption band with its maximum around 700 nm ( ε 700 nm = 1.3 (mM monomer) −1 · cm −1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) ( S = 5 2 ) in an environment of nearly axial symmetry ( g values at 7.2–6.7, 4.7–5.3 and 1.9–2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.