Purification and characterization of the blue-green rat phaeochromocytoma (PC12) tyrosine hydroxylase with a dopamine-Fe(III) complex. Reversal of the endogenous feedback inhibition by phosphorylation of serine-40

Tyrosine hydroxylase (TH) was purified from tumours of rat phaeochromocytoma (PC12) cells by a three-step purification procedure giving 30 mg of pure enzyme in 3 days. The enzyme sedimented with an s(eo,w) value of 9.2 S and revealed an apparent subunit molecular mass of 62 kDa with a minor 60 kDa component. Two-dimensional gel isoelectric focusing/electrophoresis and tryptic digestion revealed that the heterogeneity could be accounted for by limited protcolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue green colour (ε₇₀₀ = 3.1 + 0.2mM-iron^-1 cm^-1) The resonance Raman spectrum obtained with λ(excitation) = 605 nm revealed the presence of an Fc(III) catecholamine complex in the isolated enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark and Haavik (1988) J. Biol. Chem. 263, 18621 -18626]. In the rat PC12 enzyme, all of the iron present (0.53 ± 0.03 atom per subunit) seems to be chelated by the feedback inhibitors (0.49 = 0.05 mol of dopamine and 0.10 ± 0.03 mol of noradrenaline per mol of subunit). The e.p.r. spectra at 3.6 K show g-values at 7.0, 5.2 and 1.9 as observed for other catecholate-complexed enzymes. After phosphorylation of serine-40 and addition of 1-tyrosine a new rhombic (|E/D| = 0.33) e.p.r. species could be observed. Phosphorylation of serine-40 by cyclic AMP-dependent protein kinase increased the catalytic activity; depending on assay conditions, up to 80-110-fold activation could be observed when measured at high TH (i.e. high endogenous catecholamine) concentration.