Stereochemical mechanism of action for thymidylate synthase based on the X-ray structure of the covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.
The structure of the Escherichia coli thymidylate synthase (TS) covalent inhibitory ternary complex consisting of enzyme, 5-fluoro-2'-deoxyuridylate (FdUMP) and 5,10-methylene tetrahydrofolate (CH2-H4PteGlu) has been determined at 2.5 A resolution using difference Fourier methods. This complex is believed to be a stable structural analog of a true catalytic intermediate. Knowledge of its three-dimensional structure and that for the apo enzyme, also reported here, suggests for the first time how TS may activate dUMP and CH2-H4PteGlu leading to formation of the intermediate and offers additional support for the hypothesis that the substrate and cofactor are linked by a methylene bridge between C-5 of the substrate nucleotide and N-5 of the cofactor. By correlating these structural results with the known stereospecificity of the TS-catalyzed reaction it can be inferred that the catalytic intermediate, once formed, must undergo a conformational isomerization before eliminating across the bond linking C-5 of dUMP to C-11 of the cofactor. The elimination itself may be catalyzed by proton transfer to the cofactor's 5 nitrogen from invariant Asp169 buried deep in the TS active site. The juxtaposition of Asp169 and bound tetrahydrofolate in TS is remarkably reminiscent of binding geometry found in dihydrofolate reductase where a similarly conserved carboxyl group serves as a general acid for protonating the corresponding pyrazine ring nitrogen of dihydrofolate.
Agouron Pharmaceuticals, Inc. La Jolla, CA 92037.
D A
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Matthews
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Villafranca
C A
CA
Janson
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Smith
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Welsh
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Freer
D001665:Binding Sites; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005468:Fluorodeoxyuridylate / Q000378:metabolism* / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000378:metabolism*; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D011487:Protein Conformation; D013763:Tetrahydrofolates / Q000378:metabolism* / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014961:X-Ray Diffraction / Q000379:methods
D003856:Deoxyuracil Nucleotides; D005493:Folic Acid Antagonists; D008024:Ligands; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase