A stable binary complex between Leishmania major thymidylate synthase and the substrate deoxyuridylate. A...

Area of Science:

• Biochemistry
• Parasitology
• Enzymology

Background:

• Thymidylate synthase (TS) activity in Leishmania major is crucial for DNA synthesis and is part of a bifunctional TS-dihydrofolate reductase (TS-DHFR) enzyme.
• Understanding nucleotide binding to TS-DHFR is essential for developing targeted anti-parasitic therapies.

Purpose of the Study:

• To investigate the binding kinetics and characteristics of deoxyuridylate (dUMP) with Leishmania major TS-DHFR.
• To elucidate the nature of the binary complex formed between dUMP and TS-DHFR and its implications for enzyme activity.

Main Methods:

• Isolation of binary enzyme-nucleotide complexes using Sephadex G-25 chromatography and nitrocellulose filter binding.
• Kinetic analysis of dUMP binding to TS-DHFR.
• Competition experiments using inhibitors and products like 5-fluorodeoxyuridylate (FdUMP) and deoxythymidylate (dTMP).

Main Results:

• A slow-binding mechanism for dUMP to TS-DHFR was identified, forming an isolable binary complex with a dissociation constant of approximately 0.1 microM.
• The stoichiometry of binding was determined to be 1 mol dUMP per mol of dimeric TS-DHFR.
• Competition studies confirmed that dUMP, FdUMP, and dTMP bind to the same active site on the enzyme.
• The preformed dUMP-TS complex was found to be catalytically incompetent and did not inhibit enzyme activity, despite slow dUMP dissociation.

Conclusions:

• dUMP binds to a catalytically incompetent site on one subunit of the dimeric TS-DHFR.
• This binding does not affect the activity of the other enzyme subunit.
• The findings suggest a non-canonical substrate binding mechanism that may have implications for drug development against Leishmania parasites.