Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P50583 (asymmetrical)
 12,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lactobacillus casei thymidylate synthase, which employs the thiol of cysteine-198 as a covalent catalyst, was reversibly coupled to thiopropyl-Sepharose 6B through the catalytic sulfhydryl group of one of its two subunits, yielding an immobilized heterodimeric form of the enzyme possessing one free active site and one covalently modified active site. Enzyme inactivated by treatment with N-ethylmaleimide, which selectively modifies the active site cysteines but not the remaining cysteines (cys-244), failed to react with the resin. Modified assay procedures were developed and utilized to characterize the activity and ligand binding properties of this unique enzymic species. The immobilized enzyme was found to catalyze thymidylate formation at its free active site but exhibited lowered specific activity (13-fold) and kcat (16-fold) values and an increased Km for dUMP (4-fold) when compared to the native, soluble enzyme. Immobilized enzyme also formed a covalent inhibitory ternary complex with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate with a stoichiometry of 0.32 (mol FdUMP: mol enzyme), about half the predicted value of 0.6-0.7. The results of this initial study suggest that the active sites of the native enzyme dimer are asymmetrical in nature, and that the chemical status of the catalytic sulfhydryl groups may play a key role in directing communication between the subunits.
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PMID:Catalysis and ligand binding by thymidylate synthase immobilized on thiopropyl-sepharose 6B. 142 18

The crystal structure of mouse thymidylate synthase (mTS) in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands, and the other being more open (dimer CD) and thus allowing weaker binding of ligands. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound, and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed.
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PMID:Crystal structure of mouse thymidylate synthase in tertiary complex with dUMP and raltitrexed reveals N-terminus architecture and two different active site conformations. 2499 39