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Pyruvate kinases have an intrinsic and conserved decarboxylase activity

DOI: 10.1042/BJ20130790 DOI Help
PMID: 24328825 PMID Help

Authors: Wenhe Zhong (University of Edinburgh) , Hugh P. Morgan (University of Edinburgh) , Matthew W. Nowicki (University of Edinburgh) , Iain W. Mcnae (University of Edinburgh) , Meng Yuan (University of Edinburgh) , Juraj Bella (University of Edinburgh) , Paul A. M. Michels (Universite Catholique de Louvain) , Linda A. Fothergill-Gilmore (University of Edinburgh) , Malcolm D. Walkinshaw (University of Edinburgh)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal , VOL 458 (2) , PAGES 301 - 311

State: Published (Approved)
Published: March 2014
Diamond Proposal Number(s): 7613

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.

Journal Keywords: decarboxylase mechanism; enzyme evolution; oxaloacetate analogue; oxaloacetate decarboxylase; pyruvate kinase; substrate recognition

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials

Instruments: I03-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 14/09/2015 12:08

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)