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

Abstract

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 proteolysis of the 62 kDa component and the presence of covalently bound phosphate. The enzyme had a strong blue-green colour (epsilon 700 = 3.1 +/- 0.2 mM-iron-1.cm-1). The resonance Raman spectrum obtained with lambda excitation = 605 nm revealed the presence of an Fe(III)-catecholamine complex in the isolate enzyme, similar to that observed in the bovine adrenal enzyme [Andersson, Cox, Que, Flatmark & 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 L-tyrosine a new rhombic (magnitude of 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.

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation

“…VmaxjK,,,-values reported for phosphorylation of hTH in the present study are among the highest reported for any natural protein kinase substrates, and even higher than that reported for the model substrate kemptide [35]. We have recently shown that rat pheochromocytoma TH purified by conventional means contains 0.53 Fe atom/mol enzyme subunit tightly bound, and a similar amount of catecholamines [16]. Thus?…”

“…Haavik We have recently shown that catecholamines form stable complexes with hTHl and hTH2 [l I], and that bovine and rat TH are both isolated as complexes with these inhibitors [13 -161. Previous structure/activity studies on the inhibition of bovine and rat TH by catecholamines have defined some basic features of this inhibition [5,6,16,171. The interpretation of these studies is, however, complicated by interference of catecholamines already tightly bound to the isolated enzyme [lo, [13][14][15] as well as by its partial phosphorylation [14, 151.…”

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H‐NMR spectroscopy

“…Thus, the EPR measurements corroborate that the active, iron-reconstituted hTH contains iron in the ferrous form whereas, on addition of dopamine, the enzyme-bound iron is oxidized. The EPR spectrum of the high-spin ( S = 5/2) Fe(II1) in the catecholamine-enzyme complex closely matches that of tyrosine hydroxylase as isolated from bovine adrenal medulla [lo] and phaeochromocytoma cells [29] and that of phenylalanine hydroxylase in the presence of catecholamines [19,371, in which the axial type of signals have been associated with the catecholate-Fe(II1) complex. These catecholamine-enzyme complexes appear to represent inactive and biologically occurring forms of tyrosine hydroxylase [lo, 131.…”

Inactivation of phosphorylated rat tyrosine hydroxylase by ascorbate in vitro

“…These concentrations provided modest protection against inactivation at pH 7.2 (Table 3). Feedback inhibitors such as dopamine and norepinephrine form stoichiometric complexes with tyrosine hydroxylase (Anderson et al, 1992;Almas et al, 1992), and this form of the enzyme may be resistant to inhibition by ascorbate.…”

“…When the steady-state kinetic parameters are measured at pH values near 7, activation is associated with both a decrease in the K,,, for the reducing pterin substrate and an increase in the V,,,, (Daubner et al, 1992). Dopamine and norepinephrine bind tightly to tyrosine hydroxylase purified from rat PC12 cells (Anderson et al, 1992) and to purified recombinant human tyrosine hydroxylase Almas et al, 1992). Phosphorylation of these enzymes by protein kinase A decreases the affinity of the enzyme for the catecholamines and results in activation.…”