Tyrosine Hydroxylase Inactivation Following cAMP‐Dependent Phosphorylation Activation

Abstract

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the Km of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, tyrosine hydroxylase is inactivated by 2 distinct processes. Because cAMP is hydrolyzed in crude extracts by a phosphodiesterase, 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 Km for pterin co-substrate, .ltoreq. 0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent Km, .gtoreq. 1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphyorlation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), it is also inactivated. This 2nd inactivation process is irreversible and is characterized kinetically by a decreasing apparent Vmax with no change in the low apparent Km for pterin co-substrate (0.2 mM). The latter inactivation processing greatly attenuated by gel filtration which resolves a low MW inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving 2 processes: reversible phosphorylation and dephosphorylation, and an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.