A stable binary complex between Leishmania major thymidylate synthase and the substrate deoxyuridylate. A slow-binding interaction.
The thymidylate synthase (TS) activity in Leishmania major resides on the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR). We have isolated, either by Sephadex G-25 chromatography or by nitrocellulose filter binding, a binary complex between the substrate deoxyuridylate (dUMP) and TS from L. major. The kinetics of binding support a "slow binding" mechanism in which dUMP initially binds to TS in a rapid, reversible pre-equilibrium step (Kd approximately 1 microM), followed by a slow first-order step (k = 3.5 X 10(-3) s-1) which results in the isolable complex; the rate constant for the dissociation of dUMP from this complex was 2.3 X 10(-4) s-1, and the overall dissociation constant was approximately 0.1 microM. The stoichiometry of dUMP to enzyme appears to be 1 mol of nucleotide bound/mol of dimeric TS-DHFR. Binary complexes between the stoichiometric inhibitor 5-fluorodeoxyuridylate (FdUMP) and TS, and between the product deoxythymidylate (dTMP) and TS were also isolated by nitrocellulose filter binding. Competition experiments indicated that each of these nucleotides were binding to the same site on the enzyme and that this site was the same as that occupied by the nucleotide in the FdUMP-cofactor X TS ternary complex. Thus, it appeared that the binary complexes were occupying the active site of TS. However, the preformed isolable dUMP X TS complex is neither on the catalytic path to dTMP nor did it inhibit TS activity, even though the dissociation of dUMP from this complex is several orders of magnitude slower than catalytic turnover (approximately 3 s-1). The results suggest that dUMP binds to one of the two subunits of the native protein in a catalytically incompetent form which does not inhibit activity of the other subunit.
D000818:Animals; D001667:Binding, Competitive; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D007895:Leishmania tropica / Q000201:enzymology*; D011799:Quinazolines / Q000494:pharmacology; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D011799:Quinazolines; D005468:Fluorodeoxyuridylate; D013938:Thymidine Monophosphate; C031662:CB 3717; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase