Pyruvate kinase from Chlamydia trachomatis is activated by fructose-2,6-bisphosphate.
Pyruvate kinase is the final regulatory point in the catabolic Embden-Meyerhoff-Parnas pathway, which controls the carbon flux of glycolytic intermediates and regulates the level of ATP in the cell. In a previous study, we identified, cloned and sequenced pyruvate kinase from the obligate intracellular bacterium Chlamydia trachomatis and demonstrated that the enzyme was active in crude extract. Here, we report the kinetic properties of highly purified C. trachomatis pyruvate kinase. The results indicate that C. trachomatis pyruvate kinase is 53.5 kDa with a pH optima of 7.3. Kinetic studies show that C. trachomatis pyruvate kinase requires both K+ and Mg2+ ions for activity, exhibits sigmoidal kinetics with respect to phosphoenolpyruvate and Michaelis-Menten kinetics with respect to ADP. In addition, C. trachomatis pyruvate kinase is able to use alternative nucleoside diphosphates as phosphate acceptors, although it shows the greatest activity with ADP. In contrast to other bacterial pyruvate kinases that are activated by AMP, our data show that AMP, in addition to ATP and GTP, inhibits C. trachomatis pyruvate kinase. Surprisingly, unlike any other known bacterial pyruvate kinase, C. trachomatis pyruvate kinase was allosterically activated by fructose-2,6-bisphosphate, an important regulatory metabolite that has only been reported in eukaryotes.
Iliffe-Lee
Emma R
ER
Department of Medical Microbiology, University of Manitoba, 730 William Avenue, Winnipeg, Canada R3E 0W3.
McClarty
Grant
G
D000494:Allosteric Regulation; D002412:Cations / Q000378:metabolism; D002692:Chlamydia trachomatis / Q000201:enzymology*; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D005635:Fructosediphosphates / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000378:metabolism; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D002412:Cations; D005635:Fructosediphosphates; D009711:Nucleotides; D015800:Protozoan Proteins; D010728:Phosphoenolpyruvate; C027652:fructose 2,6-diphosphate; D011770:Pyruvate Kinase