Bacterial versus human thymidylate synthase: Kinetics and functionality.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Thymidylate Synthase (TSase) is crucial for DNA synthesis and highly conserved across species.
• Bacterial TSase is a model for human TSase, yet differences exist, including unique human enzyme features.
• Current TSase inhibitors are chemotherapeutics, not antibiotics, highlighting a gap in antimicrobial development.

Purpose of the Study:

• To comprehensively characterize human (hs) TSase.
• To delineate similarities and contrasts between human (hs) TSase and Escherichia coli (ec) TSase.
• To explore kinetic and functional differences for potential antibiotic drug development.

Main Methods:

• Enzyme kinetics studies comparing hsTSase and ecTSase.
• Analysis of temperature dependence of intrinsic kinetic isotope effects (KIEs).
• Investigation of substrate binding order for both enzymes.

Main Results:

• Unlike ecTSase, Mg2+ does not enhance hsTSase reaction rates.
• Kinetic isotope effects suggest a looser transition state for hydride transfer in hsTSase compared to ecTSase.
• Substrate binding is strictly ordered for ecTSase but less so for hsTSase.

Conclusions:

• Significant kinetic and functional differences exist between human and bacterial TSases.
• These differences, particularly Mg2+ independence and transition state characteristics, distinguish hsTSase.
• Understanding these distinctions may facilitate the design of novel, less toxic antibiotic agents targeting bacterial TSase.