The Two Phosphofructokinase Gene Products of Entamoeba histolytica

Abstract

Two phosphofructokinase genes have been described previously in Entamoeba histolytica. The product of the larger of the two genes codes for a 60-kDa protein that has been described previously as a pyrophosphate (PP i )-dependent enzyme, and the product of the second, coding for a 48-kDa protein, has been previously reported to be a PP i -dependent enzyme with extremely low specific activity. Here it is found that the 48-kDa protein is not a PP i -dependent enzyme but a highly active ATP-requiring enzyme (k cat ‫؍‬ 250 s ؊1 ) that binds the cosubstrate fructose 6-phosphate (Fru-6-P) with relatively low affinity. This enzyme exists in concentration-and ATP-dependent tetrameric active and dimeric inactive states. Activation is achieved in the presence of nucleoside triphosphates, ADP, and PP i , but not by AMP, P i , or the second substrate Fru-6-P. Activation by ATP is facilitated by conditions of molecular crowding. Divalent cations are not required, and no phosphoryl transfer occurs during activation. Kinetics of the activated enzyme show cooperativity with Fru-6-P (Fru-6-P 0.5 ‫؍‬ 3.8 mM) and inhibition by high ATP and phosphoenolpyruvate. The enzyme is active without prior activation in extracts of E. histolytica. The level of mRNA, the amount of enzyme protein, and the enzyme activity of the 48-kDa enzyme are about one-tenth that of the 60-kDa enzyme in extracts of E. histolytica trophozoites.Entamoeba histolytica along with a number of other parasitic protists utilizes an unusual form of phosphofructo-1-kinase (PFK) 1 in a central step in carbohydrate metabolism. This form of PFK employs inorganic pyrophosphate (PP i ) as a phosphoryl donor. Two genes for PP i -PFK have been described in E. histolytica (1-3) with a sequence identity between the two proteins of 17%. The sequence of the larger gene, which codes for a protein of ϳ60 kDa, has greater identity to the more phylogenetically advanced plant PP i -PFKs than it does to bacterial PP i -PFKs. The cDNA of this gene has been expressed in Escherichia coli and was found to have kinetic properties that were identical to those of the enzyme isolated from E. histolytica (3).

Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

“…Similarly, some residues involved in the binding of AMP in E. coli PFK, like Phe73, Ser105 and Met107, were replaced by Gly, Thr and Arg, respectively (Lopez et al 2002). These changes were also found in the PP i -dependent PFK of Entamoeba histolytica (Chi et al 2001).…”

Biochemical and Functional Characterization of PKS11, a Novel Arabidopsis Protein Kinase

“…In this study, Mn 2ϩ at concentrations from 0.25 to 2.5 mM activated PKS11T161D in the presence of 10 M ATP (Fig. 4A) (39). Mg 2ϩ was also the preferred cofactor for pyruvate kinase (40).…”

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

“…ATP-PFK activity was determined in the glycolytic direction using a continuous spectrophotometric assay according to the method of Chi et al (37) with some modifications. The assay mixture for ATP-PFK consisted of 2 ml of 100 mM HEPES, 50 mM KCl, 3 mM MgCl 2 , 1 mM EDTA, pH 7.0, buffer; 1 mM ATP; 20 mM fructose-6-phosphate; 0.15 to 0.20 mM NADH; 2 to 3 U each of aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase (Sigma-Aldrich); and 0.05% (vol/vol) Triton X-100 (ATP-PFK assay buffer).…”