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Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase
Louis T J Delbaere1, Athena M Sudom, Lata Prasad
1Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5. louis.delbaere@usask.ca
Biochimica Et Biophysica Acta
|March 17, 2004
Summary
Phosphoenolpyruvate carboxykinase (PCK) enzyme structure reveals a novel fold and active site dynamics. Structural analysis of PCK complexes elucidates the mechanism of phosphoryl transfer.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Phosphoenolpyruvate carboxykinase (PCK) is crucial for gluconeogenesis, catalyzing the conversion of oxaloacetate to phosphoenolpyruvate.
- Understanding PCK's structure and mechanism is vital for metabolic pathway research.
Purpose of the Study:
- To determine the high-resolution structures of Escherichia coli PCK.
- To elucidate the catalytic mechanism of PCK through structural analysis of enzyme-ligand complexes.
Main Methods:
- X-ray crystallography was used to determine the structures of PCK and its complexes.
- High-resolution structures were obtained at 1.8-2.0 Å.
Main Results:
- Escherichia coli PCK exhibits a unique protein fold with N- and C-terminal domains enclosing the active site.
- Complex structures revealed domain rotation, an unusual ATP conformation, and stabilization of a high-energy phosphoryl transfer state.
- The AlF3 moiety in the PCK/ADP/Mg(2+)/AlF(3) complex mimics the transition state, showing interactions with positively charged residues.
Conclusions:
- PCK possesses a novel fold and unique mononucleotide-binding fold.
- Enzyme-bound ATP adopts a strained syn conformation, facilitating phosphoryl transfer.
- Structural data supports an S(N)2-type mechanism for phosphoryl transfer and highlights the role of active site residues in catalysis.