We reported previously that, following phosphorylation by
cyclic AMP-dependent protein kinase,
tyrosine hydroxylase in rat corpus striatal extracts is inactivated in a time-dependent and apparently irreversible fashion. Removal of low molecular weight substances from these extracts by gel filtration attenuates this inactivation. We tried to determine the identity of endogenous metabolites that promote inactivation of
tyrosine hydroxylase under our experimental conditions. In the present study, we report that the reducing co-substrate
tetrahydrobiopterin and its analogues promoted this irreversible inactivation. The concentration that produced a 50% loss of activity (at 20 min) of the phosphorylated
enzyme was 0.7 microM and that for the unphosphorylated
enzyme was 420 microM. Using
enzyme purified from a rat
pheochromocytoma, we found that
tyrosine,
alpha-methyl-p-tyrosine, and a
3-iodotyrosine protected the phosphorylated
enzyme against the inactivation produced by
tetrahydrobiopterin.
Catecholamines (
dopamine,
norepinephrine,
epinephrine, and some of their analogues) also nullified inactivation. In contrast, the product of the reaction,
dihydroxyphenylalanine, failed to attenuate the inactivation process. We performed several studies to ascertain the mechanism of inhibition by
tetrahydrobiopterin. We considered the possibility that it formed reactive
free radicals that produced inhibition.
Free radical scavengers, however, failed to block the inhibition produced by
tetrahydrobiopterin.
Superoxide dismutase,
catalase, and
peroxidase also failed to protect
tyrosine hydroxylase against inactivation. Moreover, when the experiments were performed under anaerobic conditions, the inactivation process was unaffected. These results suggest that reactive oxygenated species were not required for inactivation by
tetrahydrobiopterin.