Dopamine biosynthesis is regulated by S-glutathionylation. Potential mechanism of tyrosine hydroxylast inhibition during oxidative stress.
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.
Borges
Chad R
CR
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Geddes
Timothy
T
Watson
J Throck
JT
Kuhn
Donald M
DM
D000595:Amino Acid Sequence; D000818:Animals; D003958:Diamide / Q000494:pharmacology; D004298:Dopamine / Q000096:biosynthesis*; D005978:Glutathione / Q000378:metabolism*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors*
D003958:Diamide; D014446:Tyrosine 3-Monooxygenase; D005978:Glutathione; D004298:Dopamine