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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. 1990

D A Matthews, and J E Villafranca, and C A Janson, and W W Smith, and K Welsh, and S Freer
Agouron Pharmaceuticals, Inc. La Jolla, CA 92037.

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.

UI MeSH Term Description Entries
D008024 Ligands A molecule that binds to another molecule, used especially to refer to a small molecule that binds specifically to a larger molecule, e.g., an antigen binding to an antibody, a hormone or neurotransmitter binding to a receptor, or a substrate or allosteric effector binding to an enzyme. Ligands are also molecules that donate or accept a pair of electrons to form a coordinate covalent bond with the central metal atom of a coordination complex. (From Dorland, 27th ed) Ligand
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D003856 Deoxyuracil Nucleotides Uracil nucleotides which contain deoxyribose as the sugar moiety. Deoxyuridine Phosphates,Nucleotides, Deoxyuracil,Phosphates, Deoxyuridine
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005468 Fluorodeoxyuridylate 5-Fluoro-2'-deoxyuridylate. An inhibitor of thymidylate synthetase. Formed from 5-fluorouracil or 5-fluorodeoxyuridine. 5-Fluoro-2'-Deoxyuridine-5'-Monophosphate,FdUMP,5 Fluoro 2' Deoxyuridine 5' Monophosphate
D005493 Folic Acid Antagonists Inhibitors of the enzyme, dihydrofolate reductase (TETRAHYDROFOLATE DEHYDROGENASE), which converts dihydrofolate (FH2) to tetrahydrofolate (FH4). They are frequently used in cancer chemotherapy. (From AMA, Drug Evaluations Annual, 1994, p2033) Antifolate,Antifolates,Dihydrofolate Reductase Inhibitor,Folic Acid Antagonist,Dihydrofolate Reductase Inhibitors,Folic Acid Metabolism Inhibitors,Acid Antagonist, Folic,Acid Antagonists, Folic,Antagonist, Folic Acid,Antagonists, Folic Acid,Inhibitor, Dihydrofolate Reductase,Inhibitors, Dihydrofolate Reductase,Reductase Inhibitor, Dihydrofolate,Reductase Inhibitors, Dihydrofolate
D006860 Hydrogen Bonding A low-energy attractive force between hydrogen and another element. It plays a major role in determining the properties of water, proteins, and other compounds. Hydrogen Bonds,Bond, Hydrogen,Hydrogen Bond
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D013763 Tetrahydrofolates Compounds based on 5,6,7,8-tetrahydrofolate.

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