title: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inactivated by S-sulfuration in vitro.
abstract: Hydrogen sulfide (H2S) is produced enzymatically by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), as well as other enzymes in mammalian tissues. These discoveries have led to the crowning of H2S as yet another toxic gas that serves as a gasotransmitter like NO and CO. H2S is thought to exert its biological effects through its reaction with cysteine thiols in proteins, yielding sulfurated thiol (-SSH) derivatives. One of the first proteins shown to be modified by H2S was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [1] where the S-sulfuration of the active site cysteine (Cys 152) resulted in ~7-fold increase in the activity of the enzyme. In the present study we have attempted to reproduce this result with no success. GAPDH in its reduced, or hydrogen peroxide, or glutathione disulfide, or nitrosonium oxidized forms was reacted with sulfide or polysulfides. Sulfide had no effect on reduced GAPDH activity, while polysulfides inhibited GAPDH to ~42% of control. S-sulfuration of GAPDH occurred at Cys 247 after sulfide treatment, Cys 156 and Cys 247 after polysulfide treatment. No evidence of S-sulfuration at active site Cys 152 was discovered. Both sulfide and polysulfide was able to restore the activity of glutathione disulfide oxidized GAPDH, but not to control untreated levels. Treatment of glutathione disulfide oxidized GAPDH with polysulfide also produced S-sulfuration of Cys 156. Treatment of a C156S mutant of GAPDH with sulfide and polysulfide resulted in S-sulfuration of Cys 152, which also caused a decrease and not an increase in enzymatic activity. Computational chemistry shows S-sulfuration of Cys 156 may affect the position of catalytic Cys 152, raising its pKa by 0.5, which may affect the nucleophilicity of Cys 152. The current study raises significant questions about the reported ability of H2S to activate GAPDH by the sulfuration of its active site thiol, and indicates that polysulfide is a stronger protein S-sulfurating agent than sulfide.
authors.affiliation: Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
authors.forename: Artur P
authors.initials: AP
authors.lastname: Jarosz
authors.affiliation: Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
authors.forename: Wanlei
authors.initials: W
authors.lastname: Wei
authors.affiliation: Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
authors.forename: James W
authors.initials: JW
authors.lastname: Gauld
authors.affiliation: Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
authors.forename: Janeen
authors.initials: J
authors.lastname: Auld
authors.affiliation: Department of Medical Biochemistry, Akdeniz University Medical School, 07070 Antalya, Turkey.
authors.forename: Filiz
authors.initials: F
authors.lastname: Özcan
authors.affiliation: Department of Medical Biochemistry, Akdeniz University Medical School, 07070 Antalya, Turkey.
authors.forename: Mutay
authors.initials: M
authors.lastname: Aslan
authors.affiliation: Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada. Electronic address: mutusb@uwindsor.ca.
authors.forename: Bulent
authors.initials: B
authors.lastname: Mutus
mesh_terms: D004789:Enzyme Activation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006862:Hydrogen Sulfide / Q000378:metabolism / Q000494:pharmacology; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013440:Sulfides / Q000378:metabolism / Q000494:pharmacology
chemical_list: D013440:Sulfides; C032915:polysulfide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006862:Hydrogen Sulfide
keywords: GAPDH; S-sulfuration; hydrogen sulfide; mass spectrometry; molecular dynamics; polysulfide; post-translational modification; sulfhydration