Nitration of a critical tyrosine residue in the allosteric inhibitor site of muscle glycogen phosphorylase impairs its catalytic activity.
Muscle glycogen phosphorylase (GP) is a key enzyme in glucose metabolism, and its impairment can lead to muscle dysfunction. Tyrosine nitration of glycogen phosphorylase occurs during aging and has been suggested to be involved in progressive loss of muscle performance. Here, we show that GP (in its T and R form) is irreversibly impaired by exposure to peroxynitrite, a biological nitrogen species known to nitrate reactive tyrosine residues, and to be involved in physiological and pathological processes. Kinetic and biochemical analysis indicated that irreversible inactivation of GP by peroxynitrite is due to the fast (k(inact)=3 x 10(4) M(-1) s(-1)) nitration of a unique tyrosine residue of the enzyme. Endogenous GP was tyrosine nitrated and irreversibly inactivated in skeletal muscle cells upon exposure to peroxynitrite, with concomitant impairment of glycogen mobilization. Ligand protection assays and mass spectrometry analysis using purified GP suggested that the peroxynitrite-dependent inactivation of the enzyme could be due to the nitration of Tyr613, a key amino acid of the allosteric inhibitor site of the enzyme. Our findings suggest that GP functions may be regulated by tyrosine nitration.
Dairou
Julien
J
Laboratoire de Cytophysiologie et Toxicologie Cellulaire (EA 1553), Université Paris Diderot-Paris 7, 75005 Paris, France; UFR de Biochimie, Université Paris Diderot-Paris 7, 75005, Paris, France.
Pluvinage
Benjamin
B
Laboratoire de Cytophysiologie et Toxicologie Cellulaire (EA 1553), Université Paris Diderot-Paris 7, 75005 Paris, France.
Noiran
Joseph
J
UFR de Biochimie, Université Paris Diderot-Paris 7, 75005, Paris, France.
Petit
Emile
E
Laboratoire de Cytophysiologie et Toxicologie Cellulaire (EA 1553), Université Paris Diderot-Paris 7, 75005 Paris, France.
Vinh
Joëlle
J
Laboratoire de Spectrométrie de Masse et Neuroprotéome ESPCI - CNRS UMR 7637, 75005 Paris, France.
Haddad
Iman
I
Laboratoire de Spectrométrie de Masse et Neuroprotéome ESPCI - CNRS UMR 7637, 75005 Paris, France.
Mary
Jean
J
UFR de Biochimie, Université Paris Diderot-Paris 7, 75005, Paris, France; Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement (EA 3106), Université Paris Diderot-Paris 7, 75005 Paris, France.
Dupret
Jean-Marie
JM
Laboratoire de Cytophysiologie et Toxicologie Cellulaire (EA 1553), Université Paris Diderot-Paris 7, 75005 Paris, France; UFR de Biochimie, Université Paris Diderot-Paris 7, 75005, Paris, France.
Rodrigues-Lima
Fernando
F
Laboratoire de Cytophysiologie et Toxicologie Cellulaire (EA 1553), Université Paris Diderot-Paris 7, 75005 Paris, France; UFR de Biochimie, Université Paris Diderot-Paris 7, 75005, Paris, France. Electronic address: rlima@ext.jussieu.fr.
D000494:Allosteric Regulation; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D024982:Glycogen Phosphorylase, Muscle Form / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008981:Molsidomine / Q000031:analogs & derivatives / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D020030:Nitric Oxide Donors / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry*
D020030:Nitric Oxide Donors; D030421:Peroxynitrous Acid; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D024982:Glycogen Phosphorylase, Muscle Form