Tyrosine Hydroxylase Inactivation Following cAMP‐Dependent Phosphorylation Activation

Tyrosine hydroxylase, the rate‐limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP‐dependent protein kinase (largely by decreasing the K^m of the enzyme for its pterin co‐substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho‐diesterase, cAMP‐dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent K^m for pterin co‐substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent K^m, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP‐dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent V^max with no change in the low apparent K^m for pterin co‐substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low‐molecular‐weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos‐phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.