Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis

The Biochemical Journal
Stephen L BearneJennifer E MacDonnell

Abstract

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde. Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not...Continue Reading

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Citations

May 26, 2004·Biochimica Et Biophysica Acta·Jennifer E MacDonnellStephen L Bearne
Nov 13, 2009·The Journal of Biological Chemistry·Sebastian DamerowFrançoise H Routier
Aug 27, 2010·The Journal of Biological Chemistry·Karen M KasselLee M Graves
Feb 9, 2011·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Iben LauritsenPernille Harris
Oct 30, 2004·European Journal of Biochemistry·Faylene A Lunn, Stephen L Bearne
Dec 29, 2011·Frontiers in Bioscience (Landmark Edition)·Benjamin W ArentsonDonald F Becker
Nov 30, 2010·Protein Expression and Purification·Patrick K ArthurT Kwaku Dayie
Jan 22, 2011·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Ji-Long Liu
Jun 5, 2002·Biochimica Et Biophysica Acta·Sławomir Milewski
Dec 17, 2009·Bioorganic & Medicinal Chemistry Letters·Alexander C RoyStephen L Bearne

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