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Pyrophosphate-dependent phosphofructokinase from the amoeba Naegleria fowleri, an AMP-sensitive enzyme
E Mertens1, J De Jonckheere, E Van Schaftingen
1Laboratoire de Chimie Physiologique, Université Catholique de Louvain, Belgium.
Abstract:
PPi-dependent phosphofructokinase (PPi-PFK) was detected in extracts of the amoeba Naegleria fowleri, with a specific activity of about 15-30 nmol/min per mg of protein, which was increased about 2-fold by 0.5 mM AMP. PPi-PFK was inactivated upon gel filtration and could be re-activated by incubation at 30 degrees C in the presence of AMP. N. fowleri PPi-PFK was purified more than 1100-fold to near homogeneity with a yield of about 25%. The pure enzyme had a specific activity of 65 mumol/min per mg of protein, and SDS/PAGE analysis showed a single band, of 51 kDa. Size-exclusion chromatography revealed the existence of two forms: a large one (approximately 180 kDa), presumably a tetramer, which was active, and a smaller one (approximately 45 kDa), presumably the monomer, which was inactive, but could be re-activated and converted into the large form by incubation at 30 degrees C in the presence of 0.5 mM AMP. Reactivation was also observed at 30 degrees C in the absence of AMP, particularly at higher enzyme concentration or in the presence of poly(ethylene glycol). Inactivation of the tetrameric enzyme was promoted by 0.25 M potassium thiocyanate. The enzyme displayed Km values of 10 and 15 microM for fructose 6-phosphate and PPi, respectively, in the forward reaction, and of 35 and 590 microM for fructose 1,6-bisphosphate and Pi in the backward reaction. The activity was dependent on the presence of Mg2+. AMP increased Vmax. about 2-fold without changing the affinity for the substrates; its half-maximal effect was observed at 2 microM.
Insights
The pyrophosphate-dependent phosphofructokinase (PPi-PFK) enzyme was identified and purified from Naegleria fowleri. AMP and temperature influence its activity and structure, suggesting regulatory roles.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Naegleria fowleri is an opportunistic freshwater amoeba.
- Phosphofructokinase (PFK) is a key enzyme in glycolysis.
- PPi-PFK utilizes pyrophosphate instead of ATP as a phosphate donor.
Purpose of the Study:
- To detect and characterize the pyrophosphate-dependent phosphofructokinase (PPi-PFK) in Naegleria fowleri.
- To investigate the enzyme's properties, including its activity, stability, and response to activators and inhibitors.
- To elucidate the enzyme's quaternary structure and its relationship with activity.
Main Methods:
- Enzyme assays to measure PPi-PFK activity.
- Protein purification techniques, including gel filtration and ion-exchange chromatography.
- SDS-PAGE and size-exclusion chromatography for molecular weight and quaternary structure determination.
- Kinetic analysis to determine Km and Vmax values.
Main Results:
- PPi-PFK was detected in N. fowleri extracts and purified over 1100-fold.
- The purified enzyme (51 kDa monomer) exists in active tetrameric (~180 kDa) and inactive monomeric (~45 kDa) forms.
- Activity and tetramer formation were enhanced by AMP and incubation at 30°C.
- Kinetic parameters (Km for fructose 6-phosphate and PPi) and Mg2+ dependence were determined.
- AMP increased Vmax approximately 2-fold without altering substrate affinity.
Conclusions:
- N. fowleri possesses an AMP-regulated PPi-PFK.
- The enzyme's activity is modulated by its quaternary structure, which is sensitive to temperature and AMP.
- The findings provide insights into the metabolic regulation of this parasitic amoeba.