Spectroscopic studies of phosphoenolpyruvate carboxykinase from Mycobacterium tuberculosis

Lucie Bednárová, Iva Machová, Jan Snášel, Jiří Dostál, Martin Hubálek and Iva Pichová

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic


Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. MTb Pck contains nine cysteine residues that might co-determine the redox state and conformation of Pck under different conditions. Structural alignment showed that Cys-273, located within the putative P loop of MTb Pck, is probably the hyperreactive cysteine residue, which is typical for the GTP-dependent Pck family and coordinates binding of Mn2+ in the active site.[1] The minor structural changes of Pck in the absence or in the presence of reducing agents were reported and the presence of one disulfide bridge was suggested.[2]

We present the spectroscopic study of the Pck and its mutants C119S and C198S to characterize this disulfide bridge. The mutation sites were chosen with respect to detail crystal structure analysis of Pck and represent the cysteines, which might form disulfide bond. Circular dichroism and Raman spectroscopy supported by mass spectroscopy was used for this purpose.


1.             Holyoak, T., Sullivan, S. M., and Nowak, T. Biochemistry 45, (2006) 8254–8263

2.           Machová I, Snášel J, Zimmermann M, Laubitz D, Plocinski P, Oehlmann W, Singh M, Dostál J, Sauer U, Pichová I.J Biol Chem, 289(19): 201413066-78.


Supported by NPU LO 1302 from Ministry of Education.