Abstract
Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inhibited by the sulfhydryl oxidant diamide in a concentration-dependent manner. The inhibitory effect of diamide on TH catalytic activity is enhanced significantly by GSH. Treatment of TH with diamide in the presence of [(35)S]GSH results in the incorporation of (35)S into the enzyme. The effect of diamide-GSH on TH activity is prevented by dithiothreitol (DTT), as is the binding of [(35)S]GSH, indicating the formation of a disulfide linkage between GSH and TH protein cysteinyls. Loss of TH catalytic activity caused by diamide-GSH is partially recovered by DTT and glutaredoxin, whereas the disulfide linkage of GSH with TH is completely reversed by both. Treatment of intact PC12 cells with diamide results in a concentration-dependent inhibition of TH activity. Incubation of cells with [(35)S]cysteine, to label cellular GSH prior to diamide treatment, followed by immunoprecipitation of TH shows that the loss of TH catalytic activity is associated with a DTT-reversible incorporation of [(35)S]GSH into the enzyme. A combination of matrix-assisted laser desorption/ionization/mass spectrometry and liquid chromatography/tandem mass spectrometry was used to identify the sites of S-glutathionylation in TH. Six cysteines (177, 249, 263, 329, 330, and 380) of the seven cysteine residues in TH were confirmed as substrates for modification. Only Cys-311 was not S-glutathionylated. These results establish that TH activity is influenced in a reversible manner by S-glutathionylation and suggest that cellular GSH may regulate dopamine biosynthesis under conditions of oxidative stress or drug-induced toxicity.
Highlights
From the ‡Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, the §Department of Psychiatry and Behavioral Neurosciences and the ʈCenter for Molecular Medicine and Genetics, Wayne State University School of Medicine, and the ¶John D
It has been proposed that the reductions in Tyrosine hydroxylase (TH) function that are seen under conditions that damage DA neurons reflect an attack on the protein by reactive oxygen or nitrogen species [45]
TH is slightly inhibited by the specific thiol oxidant diamide [46] and this inhibition of enzyme activity is significantly enhanced by GSH
Summary
Vol 277, No 50, Issue of December 13, pp. 48295–48302, 2002 Printed in U.S.A. POTENTIAL MECHANISM OF TYROSINE HYDROXYLASE INHIBITION DURING OXIDATIVE STRESS*□S. Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inhibited by the sulfhydryl oxidant diamide in a concentration-dependent manner. We report presently that tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the synthesis of the neurotransmitter dopamine, is modified by S-glutathionylation and this postradish peroxidase; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; RP, reverse phase; ANOVA, analysis of variance. Regulation of Tyrosine Hydroxylase by S-Glutathionylation translational modification results in a reduction in TH catalytic function
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