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Active-site mutants altering the cooperativity of E. coli phosphofructokinase
1MRC Laboratory of Molecular Biology, Cambridge, UK.
Nature
|February 8, 1990
Summary
Three key arginines in phosphofructokinase are crucial for substrate binding, catalysis, and enzyme cooperativity. Mutations reveal their specific roles in allosteric regulation and signal transmission within the enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Phosphofructokinase (PFK) is a key allosteric enzyme regulating glycolysis.
- Understanding PFK's structure-function relationship is vital for metabolic control insights.
Purpose of the Study:
- To elucidate the roles of specific arginine residues in phosphofructokinase (PFK) activity, substrate binding, and allosteric regulation.
- To investigate how these arginines contribute to signal transmission between enzyme subunits.
Main Methods:
- X-ray crystallography to determine high- and low-activity states of PFK.
- Site-directed mutagenesis of key arginine residues (R162, R243, R72) to serine.
- Enzyme kinetics assays to assess substrate binding, catalytic activity (kcat), and cooperativity.
Main Results:
- Arginines 162 and 243 are critical for fructose 6-phosphate binding and cooperativity, with mutations reducing binding and cooperativity but not kcat.
- Arginine 72 interacts with both substrates and product, and its mutation impairs catalysis, cooperativity, and substrate binding.
- Mutant kinetics revealed distinct effects: Ser72 mutation altered substrate kinetics, while Ser162 and Ser243 mutations retained sigmoidal kinetics in the reverse reaction, indicating roles in cooperativity.
Conclusions:
- Each of the three studied arginines plays a distinct and essential role in PFK's allosteric regulation.
- These residues are key mediators of substrate binding, catalysis, and the propagation of allosteric signals across enzyme subunits.