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Mutagenesis, structure and function studies of thymidylate synthase

¹Department of Biochemistry and Biophysics, University of California, San Francisco 94143.

Nucleic Acids Symposium Series

|January 1, 1993

Summary

Thymidylate synthase (TS) studies reveal few essential amino acids and a key role for water. A single mutation alters enzyme specificity, impacting its catalytic function.

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Area of Science:

Biochemistry
Enzymology
Molecular Biology

Background:

Thymidylate synthase (TS) is crucial for DNA synthesis.
The enzyme's mechanism and structure are largely understood.
Amino acid residue functions are typically studied via mutagenesis.

Purpose of the Study:

Investigate the functional roles of amino acid residues in thymidylate synthase.
Explore the catalytic mechanism and substrate specificity of TS.
Identify key residues and factors influencing enzyme activity.

Main Methods:

Replacement set mutagenesis to probe amino acid function.
Detailed analysis of enzyme variants.
Structure-function relationship studies.

Main Results:

Few amino acids in TS are strictly essential for function.
Water may act as a general acid/base catalyst in the reaction.
A single mutation (Asn229Asp) switches TS specificity from dUMP to dCMP methylation.

Conclusions:

The catalytic mechanism of TS is more flexible than previously thought.
Water plays a significant catalytic role in thymidylate synthase.
Specific mutations can dramatically alter enzyme substrate specificity.

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