Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions

Abstract

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to dopa. cAMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. Short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg .cntdot. ATP, cAMP) increased enzyme activity 2- to 10-fold over control as measured during a subsequent 15 min assay. Pre-incubation under phosphorylating conditions for longer periods (30 min) resulted in a loss of activity to levels equal to or below that of the control enzyme. The addition of purified bovine brain protein kinase catalytic subunit and Mg .cntdot. ATP enhanced activation and increased the rate of inactivation. To demonstrate that inactivation was not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme was reactivated. The protein kinase inhibitor protein decreased the activation process and prevented inactivation of the enzyme to below control values. The sedimentation coefficient was not changed by phosphorylation conditions (S = 8.8 .+-. 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor was reduced (0.86 mM, control; 0.32 mM, activated), it was reduced in the inactivated form (0.38 mM). The Ki for dopamine was increased from 4.5 .mu.M for the control to 28 .mu.M for the activated enzyme. The inactivated form of the enzyme exhibited a Ki of 10 .mu.M. Removal of catecholamines by gel filtration failed to alter activity and the apparent cofactor Km. The activated and the inactivated states persisted following gel filtration. The activation-inactivation process apparently is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. A coupled decarboxylase assay for resolving labeled dopamine from the precursors tyrosine and dopa by low-voltage paper electrophoresis is described.