Molecular basis of the unusual catalytic preference for GDP/GTP in Entamoeba histolytica 3-phosphoglycerate kinase
2009
Encalada, Rusely | Rojo-Domínguez, Arturo | Rodríguez-Zavala, José S. | Pardo, Juan P. | Quezada, Héctor | Moreno Sánchez, Rafael | Saavedra, Emma
Phosphoglycerate kinase (EC 2.7.2.3) catalyzes reversible phosphoryl transfer from 1,3-bisphosphoglycerate to ADP to synthesize 3-phosphoglycerate and ATP during glycolysis. Phosphoglycerate kinases from several sources can use GDP/GTP as alternative substrates to ADP/ATP; however, the maximal velocities (Vm) reached with the guanine nucleotides are ~ 50% of those displayed with the adenine nucleotides. By contrast, Entamoeba histolytica phosphoglycerate kinase (EC 2.7.2.10) is the only reported phosphoglycerate kinase displaying higher activity with GDP/GTP and lower affinities for the adenine nucleotides. To elucidate the molecular basis of the Entamoeba histolytica phosphoglycerate kinase selectivity for GDP/GTP, a conformational analysis was carried out on a homology model based on crystallographic structures of yeast and pig phosphoglycerate kinases. Some amino acid residues involved in the purine ring binding site not previously described were detected. Accordingly, Y239, E309 and V311 were replaced by site-directed mutagenesis in the Entamoeba histolytica phosphoglycerate kinase gene for the corresponding amino acid residues present in the adenine nucleotide-dependent phosphoglycerate kinases and the recombinant proteins were purified. Kinetic analysis of the enzymes showed that the single mutants Y239F, E309Q, E309M and V311L increased their catalytic efficiencies (Vm/Km) with ADP/ATP as a result of both, increased Vm and decreased Km values. Furthermore, a higher catalytic efficiency in the double mutant Y239F/E309M was achieved, which was mainly due to an increased affinity for ADP/ATP with a concomitant diminished affinity for GDP/GTP. The main Entamoeba histolytica phosphoglycerate kinase amino acid residues involved in the selectivity for guanine nucleotides were thus identified.
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