Catalysis and ligand binding by thymidylate synthase immobilized on thiopropyl-sepharose 6B.
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.
Bradshaw
T P
TP
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208.
Dunlap
R B
RB
D004800:Enzymes, Immobilized; D005033:Ethylmaleimide / Q000494:pharmacology; D005468:Fluorodeoxyuridylate / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014238:Trichloroacetic Acid / Q000494:pharmacology
D004800:Enzymes, Immobilized; D005468:Fluorodeoxyuridylate; D014238:Trichloroacetic Acid; C035872:thiopropyl-sepharose; D012685:Sepharose; D013940:Thymidylate Synthase; D005033:Ethylmaleimide