Tyrosine hydroxylase activity is regulated through the modification of the 176th cysteine residue

Abstract

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine (DA), and the regulation of its activity is important for DA homeostasis. In this study, we focused on the modification of TH through a cysteine residue. We found that incubation with N-ethylmaleimide (NEM), a cysteine modification reagent, inactivated TH. The responsible cysteine was identified as Cys176 of human TH with recombinant mutant proteins. We further examined how NEM modification was affected by the states of TH. DA binding, a feedback inhibition mechanism of TH, delayed the modification and inactivation of TH by NEM. In contrast, the S40E mutant, which mimics the phosphorylation of Ser40 that suppresses DA binding and is thus considered as an active state of TH, did not affect modification and inactivation. These results suggest that the modification of Cys176 can inhibit even phosphorylated active TH. In addition, we found that DA oxides, which are generated by oxidative stress in dopaminergic neurons, reacted with TH through Cys176 and inhibited its activity, similar to NEM. These results suggest that the modification of Cys176 of TH could be involved in the mechanisms of neurotoxicity caused by DA oxides.

Keywords: Cysteine modification; Dopamine; Dopamine oxides; N-ethylmaleimide; Parkinson's disease; Tyrosine hydroxylase.