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Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. a phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the chage in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary hitory, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs. [ABSTRACT FROM AUTHOR]

A study has been performed in order to try to explain some anomalies which are observed in aminoacylation reactions: the extent of aminoacylation often depends upon the experimental conditions, such as the enzyme concentration or the ionic strength. We have demonstrated that these anomalies can be explained, in the case of yeast tRNAIIVal—valyl-tRNA synthetase system, by the existence of two reactions occurring beside the aminoacylation reaction: the spontaneous deacylation of the valyl-tRNAVal and a valyl-tRNA synthetase-catalyzed deacylation of the valyl-tRNAVal. Consequently, any aminoacylation plateau merely reflects the existence of an equilibrium between the aminoacylation reaction and the deacylation reactions. Sometimes the aminoacylation kinetics do not present a plateau but a maximum value followed by a decrease with time. This can be explained by an additional reaction: a valyl-tRNA synthetase-catalyzed degradation of ATP into AMP and PPi in the presence of tRNAVal and of valine. This reaction leads to an accumulation of AMP and PPi favouring the reverse reaction of the aminoacylation. The kinetic constants of these reactions have been determined. Knowing these constants, it is possible to calculate theoretical aminoacylation plateau values which are in good agreement with the experimental ones. These interpretations can probably be extended to all the tRNA aminoacylation systems. [ABSTRACT FROM AUTHOR]

The leueyl-tRNA synthctase from Bacillus stearothermophilus has been purified 350-fold. The enzyme is stable when stored at 4° in the presence of sulfhydryl protecting compounds. The Km values for leucine and ATP are respectively 240 and 0.84 μM. The ATP-PP1 exchange and the aminoacylation reaction satisfy the Arrhenius law up to 45°. After thermal denaturation the enzyme has been partially renatured. [ABSTRACT FROM AUTHOR]

The effect of acetylation and of sulfhydryl blocking agents on phosphofructokinase from rabbit muscle has been studied. Treatment of phosphofructokinase with N-acetylimidazole led to rapid loss of its allosteric function, as measured by the ability of AMP to stimulate the ATP-inhibited enzyme. The catalytic activity was far less sensitive to acetylation. The loss of allosteric function on acetylation was due to the fact that the modified enzyme was insensitive to ATP inhibition. The presence of ATP during acetylation prevented the loss of allosteric activity. The results indicate that tyrosine residues may play an important role in the inhibitory binding sites of ATP. Incubation of phosphofructokinase with a 50-fold excess of para-chloromercuribenzoate led to complete inactivation of the enzyme, an effect which could be reversed by the addition of excess thiol. Sulfhydryl blocking had no effect on the ability of AMP to stimulate the ATP- inhibited enzyme. The results confirm that sulfhydryl groups are closely associated with the catalytic site of phosphofructokinase, and demonstrate that they are not involved in the inhibition of the enzyme by ATP and its reversal by AMP. [ABSTRACT FROM AUTHOR]