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Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation

DOI: 10.1042/BCJ20180556 DOI Help

Authors: Alice Rose Mitchell (University of Edinburgh) , Meng Yuan (University of Edinburgh) , Hugh P. Morgan (University of Edinburgh) , Iain W. Mcnae (University of Edinburgh) , Elizabeth A. Blackburn (University of Edinburgh) , Thierry Lebihan (University of Edinburgh) , R. A. Homem (University of Edinburgh) , M. Yu (University of Edinburgh) , Gary J. Loake (Univerisity of Edinburgh) , Paul A. Michels (University of Edinburgh) , Martin A. Wear (University of Edinburgh) , Malcolm D. Walkinshaw (University of Edinburgh)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal

State: Published (Approved)
Published: September 2018
Diamond Proposal Number(s): 18515

Abstract: We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation and that these modifications regulate enzyme activity by preventing formation of the active tetrameric form. The biotin switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358 and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80%, 100%, and 140% activity, respectively compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast the Cys326Ser mutant exists predominantly in monomeric form. A biotin switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer-tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

Journal Keywords: pyruvate kinase; nitrosation; enzyme activity; redox signalling

Subject Areas: Biology and Bio-materials


Instruments: I02-Macromolecular Crystallography