title: Pyruvate kinases have an intrinsic and conserved decarboxylase activity.
abstract: The phosphotransfer mechanism of PYKs (pyruvate kinases) has been studied in detail, but the mechanism of the intrinsic decarboxylase reaction catalysed by PYKs is still unknown. 1H NMR was used in the present study to follow OAA (oxaloacetate) decarboxylation by trypanosomatid and human PYKs confirming that the decarboxylase activity is conserved across distantly related species. Crystal structures of TbPYK (Trypanosoma brucei PYK) complexed with the product of the decarboxylase reaction (pyruvate), and a series of substrate analogues (D-malate, 2-oxoglutarate and oxalate) show that the OAA analogues bind to the kinase active site with similar binding modes, confirming that both decarboxylase and kinase activities share a common site for substrate binding and catalysis. Decarboxylation of OAA as monitored by NMR for TbPYK has a relatively low turnover with values of 0.86 s-1 and 1.47 s-1 in the absence and presence of F26BP (fructose 2,6-bisphosphate) respectively. Human M1PYK (M1 isoform of PYK) has a measured turnover value of 0.50 s-1. The X-ray structures explain why the decarboxylation activity is specific for OAA and is not general for α-oxo acid analogues. Conservation of the decarboxylase reaction across divergent species is a consequence of piggybacking on the conserved kinase mechanism which requires a stabilized enol intermediate.
authors: *Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
authors: Wenhe
authors: W
authors: Zhong
authors: *Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
authors: Hugh P
authors: HP
authors: Morgan
authors: *Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
authors: Matthew W
authors: MW
authors: Nowicki
authors: *Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
authors: Iain W
authors: IW
authors: McNae
authors: *Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
authors: Meng
authors: M
authors: Yuan
authors: †Edinburgh Biomolecular NMR Unit, Joseph Black Building, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, U.K.
authors: Juraj
authors: J
authors: Bella
authors: Paul A M
authors: PA
authors: Michels
authors: *Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
authors: Linda A
authors: LA
authors: Fothergill-Gilmore
authors: *Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
authors: Malcolm D
authors: MD
authors: Walkinshaw
mesh_terms: D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003653:Decarboxylation / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology
chemical_list: D011770:Pyruvate Kinase