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Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase
Published on: December 19, 2011
Acetyl-CoA enolization in citrate synthase: a quantum mechanical/molecular mechanical (QM/MM) study
1Physical and Theoretical Chemistry Laboratory, Oxford University, United Kingdom.
Proteins
|January 1, 1997
Summary
Citrate synthase stabilizes the acetyl-CoA enolate intermediate, crucial for efficient citrate formation. This study clarifies the reaction mechanism, identifying Asp-375 as the catalytic base.
Area of Science:
- Biochemistry
- Enzymology
- Computational Chemistry
Background:
- Citrate synthase catalyzes citrate formation via acetyl-CoA and oxaloacetate.
- The reaction's rapid rate is puzzling due to the instability of the proposed acetyl-CoA enolate intermediate.
- Alternative mechanisms involving acetyl-CoA enol or low-barrier hydrogen bonds have been suggested.
Purpose of the Study:
- To investigate the mechanism of acetyl-CoA enolization in citrate synthase.
- To determine the stability and role of reaction intermediates.
- To elucidate the function of active site residues, specifically Asp-375 and His-274.
Main Methods:
- Quantum mechanical/molecular mechanical (QM/MM) calculations.
- Analysis of acetyl-CoA enolization pathway.
- Assessment of intermediate stability and active site interactions.
Main Results:
- QM/MM calculations support Asp-375 as the catalytic base.
- The acetyl-CoA enolate is more stable than the enol at the active site.
- His-274 is found to be neutral and stabilizes the enolate via hydrogen bonds, along with a water molecule.
- Conditions for a low-barrier hydrogen bond are not met.
Conclusions:
- The acetyl-CoA enolate intermediate is stabilized within the citrate synthase active site.
- Asp-375 acts as the catalytic base, facilitating the reaction.
- The enolate's stability is essential for the efficient condensation reaction, clarifying the enzyme's mechanism.
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