EXPRESSION AND CHARACTERIZATION OF RECOMBINANT PYRUVATE PHOSPHATE DIKINASE FROM ENTAMOEBA-HISTOLYTICA

The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis. It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate d onor. Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosp hate (PPi)-dependent enzyme, substitutes pyruvate kinase present in hu mans. We previously cloned and sequenced the gene that codifies for PP DK in E. hisyolytica. We now report its expression in a bacterial syst em and its purification to 98% homogeneity. We determined its K-m for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 mu M, respectively). Unlike PPDK from maize and bacteria and pyruvate kinase from other ce lls, EhPPDk is dependent on divalent cations but does not require mono valent cations for activity. The enzyme has an optimum pH of 6.0, it i s labile to low temperatures and has a tetrameric structure. Since EhP PDK is a PPi-dependent enzyme, we also tested the effect of some pyrop hosphate analogs as inhibitors of activity. Studies on the function an d structure of this enzyme may be important for therapeutic research i n several parasitic diseases, since it has no counterpart in humans. ( C) 1998 Elsevier Science B.V.