The pyrophosphate-dependent phosphofructokinase (PP i -PFK) of Entamoeba histolytica displays a millionfold preference for inorganic pyrophosphate (PP i ) over ATP (calculated as the ratio of k cat /K m ). The introduction of a single mutation by site-directed mutagenesis changes its preference from PP i to ATP. The single mutant has an 8-fold preference for ATP whereas a related double mutant shows a preference exceeding 10,000-fold. The results suggest the presence of a latent nucleotide binding site aligned for a catalytic role in PP i -PFK. It is proposed that the ancestral PFK was an ATP-dependent enzyme and that PP i -PFKs are a later evolving adaptation.In the mid-1960s Calvin and Lipmann (1, 2) independently first suggested a role for pyrophosphate as a high energy bond donor in the primeval earth. They proposed that the reactions found in primitive life forms evolved from prebiotic systems and that contemporary primitive organisms could retain the ability to employ inorganic pyrophosphate (PP i ) 1 as a high energy compound. Suggestions that PP i could serve as an energy source found later support in the discovery of a number of instances in which polyphosphates or pyrophosphate could substitute for ATP in reactions of glycolysis: glucokinase, pyruvate kinase, phosphoglycerate kinase, and phosphofructokinase (see Ref. 3 for review). In most instances these enzymes were found in anaerobic bacteria and presumptive primitive protists, such as Giardia and Entamoeba, supporting the idea that the kinases employed ancient mechanisms. Of the PP i -dependent enzymes, phosphofructokinase (PFK) has been studied most extensively. Sequence data have shown the pyrophosphate-dependent PFK (PP i -PFK) to be homologous to the ATP-dependent phosphofructokinase (ATP-PFK), but with amino acid identities being less than 35% even when all positions that are not shared among all PFKs are eliminated (4). In a recent study of a PP i -PFK from the archaeon, Thermoproteus tenax, Siebers et al. (5) suggested that the archeal PP i -PFK represents the most ancient lineage of PFKs. In the current study we demonstrate the presence of a latent nucleotide binding site in the PP i -PFK of Entamoeba histolytica, suggesting that the nucleotide-dependent enzyme is the more primitive phosphofructokinase. EXPERIMENTAL PROCEDURESThe plasmid bearing the E. histolytica PP i -PFK gene cloned into the prokaryotic expression plasmid pALTER-Ex1 (Promega) at the NdeI and XbaI restriction sites has been described previously (6). Site-directed mutagenesis was performed by using the Altered Sites II In Vitro Mutagenesis System (Promega). Recombinant wild type and mutant plasmids were transformed into DF1020 (pro-82, ⌬pfk201, recA56, ⌬(rha-pfkA)200, endA1, hsdR17, supE44) Escherichia coli and plated onto Luria broth agar plates (100 g/ml ampicillin). Freshly transformed single colonies were inoculated into Luria broth medium (100 g/ml ampicillin) and grown at 37°C until A 600 reached 0.6. Isopropyl-␤-D-thiogalactopyranoside was then added to a final c...

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).

Freshwater fish face a variety of spatiotemporal thermal challenges throughout their life. On a broad scale, temperature is an important driver of physiological, behavioural and ecological patterns and ultimately affects populations and overall distribution.These broad patterns are partly underpinned by the small-scale local effects of temperature on individuals within the population. Climate change is increasing the range of daily thermal variation in most freshwater ecosystems, altering behaviour and performance of resident fishes. The aim of this review is understanding how daily thermal variation in temperate rivers affects individual fish physiology, behaviour and overall performance. The following are highlighted in this study: (a) the physical characteristics of rivers that can either buffer or exacerbate thermal variability, (b) the effects of thermal variability on growth and metabolism, (c) the approaches for quantifying thermal variation and thermal stress and (d) how fish may acclimatize or adapt to our changing climate.

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