Browsing by Author "Fejzullahi, Ardit"

Membrane-bound pyrophosphatases (M-PPases) catalyse the reversible hydrolysis of pyrophosphate into two inorganic phosphate molecules. This hydrolysis is coupled to the transport of protons and/or sodium ions across the biological membrane, generating an electrochemical gradient, which can be utilized by the host organism as an energy source under different stress conditions. The essential physiological roles of M-PPases in agriculturally relevant plants and various human pathogens, such as Bacteroides fragilis, Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei, make them an important research area. Despite the biochemical studies conducted with several M-PPases and structural characterization of Thermotoga maritima and Vigna radiata M-PPases, there remain several questions regarding the biochemistry and catalytic mechanism of M-PPases, one being the basis of K+ dependence. Mutational studies have suggested that a single residue at position 495 is the sole determinant of K+ dependence in M-PPases. In K+-dependent M-PPases this residue is an alanine and a K+ ion binding in close proximity of it, has been shown of having stimulatory effect on K+- dependent M-PPases by increasing the maximal rate of pyrophosphate hydrolysis. In K+-independent M-PPases, a lysine residue resides at 495 position, where the ε-NH3+ group of the lysine residue has been suggested, based on structural modelling and AlaàLys (A495K) mutational studies, to structurally and functionally mimic the activating function of K+ in K+-dependent M-PPases. In order to provide structural basis to this, in this study, a M-PPase belonging to a hyperthermophilic deep- sea bacterium T. maritima containing a single A495K point mutation was expressed in Saccharomyces cerevisiae, solubilized using the high-temperature (“Hot-solve”) method, purified with metal affinity chromatography and structurally characterized using X-ray crystallography. As suggested, the ε-NH3+ group of the introduced lysine was shown to occupy the same space and form the same interactions at the active site as the K+ ion in K+-dependent M-PPases. However, these structural findings were in contradiction with the conducted hydrolytic activity assay which showed that the A495K mutation, besides abolishing K+ dependence, significantly reduced the overall activity compared to the wild type by ~2-10-fold, depending on the K+ concentration. These findings suggest that additional factor(s) besides the 495 residue determine the K+ dependence in M-PPases.