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Substrate connectivity effects in the transition state for cytidine deaminase
1Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599, USA.
Biochemistry
|August 26, 1998
Summary
Escherichia coli cytidine deaminase binds cytidine more effectively than its components, cytosine and ribofuranose. The glycosidic bond significantly enhances binding affinity, crucial for enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Escherichia coli cytidine deaminase is a key enzyme in nucleotide metabolism.
- Understanding substrate binding is crucial for enzyme mechanism elucidation.
Purpose of the Study:
- To compare the binding properties of cytidine and its fragments (cytosine, ribofuranose) to E. coli cytidine deaminase.
- To investigate the role of the glycosidic bond in substrate binding and enzyme catalysis.
Main Methods:
- Enzyme kinetics studies (kcat/Km) were performed.
- Competitive inhibition assays were used to determine binding affinities (Kd, Ki).
- Ligands and their fragments were used as substrates and inhibitors.
Main Results:
- Cytosine is a very slow substrate compared to cytidine, despite similar active site entry.
- The binding affinity of cytidine is significantly higher (9.5 kcal/mol) than the sum of its parts.
- The glycosidic bond contributes substantially (9.9 kcal/mol) to the binding energy of transition state analogue inhibitors.
Conclusions:
- The intact cytidine molecule binds much more favorably than its individual components.
- The glycosidic bond plays a critical role in the binding energy and catalytic efficiency of cytidine deaminase.
- Enzyme active site structure optimizes binding of the complete substrate for efficient catalysis.