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

Alice Rose Mitchell, Meng Yuan, Hugh P Morgan, Iain W McNae, Elizabeth A Blackburn, Thierry LeBihan, R A Homem, M Yu, Gary J Loake, Paul A Michels, Martin A Wear, Malcolm D Walkinshaw

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)3275-3291
Number of pages17
JournalBiochemical Journal
Volume475
Issue number20
Early online date25 Sep 2018
DOIs
Publication statusPublished - 31 Oct 2018

Keywords

  • enzyme activity
  • nitrosation
  • pyruvate kinase
  • redox signalling

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