Peroxynitrite-induced nitration of tyrosine hydroxylase - Identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis
Kuhn, DM; Sadidi, M; Liu, XL; Kreipke, C; Geddes, T; Borges, C; Watson, JT
Tyrosine hydroxylase (TH), the initial and rate-limiting
enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The
sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted
laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8
proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-
flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH,
showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of
one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH.
The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to
phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH
activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated
by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants
Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of
control) after exposure to peroxy-nitrite, and the triple mutant Y423F,Y428F,Y432F was not a
substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all
tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that
specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite
resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432)
as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be
critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient
for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite
is associated instead with oxidation of cysteine residues.