A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

Hugh P Morgan, Martin J Walsh, Elizabeth A Blackburn, Martin A Wear, Matthew B Boxer, Min Shen, Henrike Veith, Iain W McNae, Matthew W Nowicki, Paul A M Michels, Douglas S Auld, Linda A Fothergill-Gilmore, Malcolm D Walkinshaw

Research output: Contribution to journalArticlepeer-review


PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.
Original languageEnglish
Pages (from-to)67-72
Number of pages6
JournalBiochemical Journal
Issue number1
Publication statusPublished - 15 Nov 2012


  • Animals
  • High-Throughput Screening Assays
  • Saccharin
  • Recombinant Proteins
  • Models, Molecular
  • Arginine
  • Benzoates
  • Humans
  • Pyruvate Kinase
  • Suramin
  • Catalytic Domain
  • Lysine
  • Enzyme Inhibitors
  • Protein Binding
  • Structure-Activity Relationship
  • Adenosine Diphosphate
  • Conserved Sequence
  • Isoenzymes
  • Crystallography, X-Ray
  • Adenosine Triphosphate
  • Leishmania mexicana
  • Species Specificity
  • Protein Conformation


Dive into the research topics of 'A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase'. Together they form a unique fingerprint.

Cite this