Dopamine biosynthesis is regulated by S-glutathionylation. Potential mechanism of tyrosine hydroxylase inhibition during oxidative stress

Chad Borges, Timothy Geddes, J. Throck Watson, Donald M. Kuhn

Research output: Contribution to journalArticle

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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 [35S]GSH results in the incorporation of 35S into the enzyme. The effect of diamide-GSH on TH activity is prevented by dithiothreitol (DTT), as is the binding of [35S]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 [35S]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 [35S]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.

Original languageEnglish (US)
Pages (from-to)48295-48302
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number50
DOIs
StatePublished - Dec 13 2002
Externally publishedYes

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Oxidative stress
Biosynthesis
Tyrosine 3-Monooxygenase
Dopamine
Oxidative Stress
Diamide
Dithiothreitol
Cysteine
Catalyst activity
Disulfides
Mass spectrometry
Enzymes
Glutaredoxins
PC12 Cells
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Liquid chromatography
Tandem Mass Spectrometry
Drug-Related Side Effects and Adverse Reactions
Immunoprecipitation
Oxidants

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dopamine biosynthesis is regulated by S-glutathionylation. Potential mechanism of tyrosine hydroxylase inhibition during oxidative stress. / Borges, Chad; Geddes, Timothy; Watson, J. Throck; Kuhn, Donald M.

In: Journal of Biological Chemistry, Vol. 277, No. 50, 13.12.2002, p. 48295-48302.

Research output: Contribution to journalArticle

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