Characterization of a novel form of thymidylate synthase: a heterodimer isolated after specific chemical modification of the immobilized native enzyme.
A novel approach, utilizing covalent chromatography and selective chemical modification, is described for application in studying subunit interactions involved in the catalytic and regulatory mechanisms of certain oligomeric proteins. The specific objective was to prepare and characterize heterodimeric form of thymidylate synthase which would serve as a model for an intermediate stage of the catalytic mechanism in which the active-site cysteine of one subunit would be engaged in covalent catalysis while that of the other subunit would exist in the free sulfhydryl or thiolate anion form. Dimeric Lactobacillus casei thymidylate synthase was subjected to covalent chromatography on thiopropyl Sepharose 6B resin under conditions in which a mixed disulfide linkage was formed with the catalytic sulfhydryl group of just one of the two subunits. Specific chemical modification of the remaining essential sulfhydryl group of the immobilized group enzyme with N-ethylmalemide, followed by cleavage and elution with buffer containing 2-mercaptoethanol, yielded the desired soluble heterodimeric form of the enzyme. The specific activity of this unique form of the enzyme (1.55 units/mg) was approximately 60% that of native protein (2.61 units/mg). Gel electrophoretic analysis of the heterodimeric enzyme, incubated in the presence of FdUMP and 5,10-methylenetetrahydrofolate (CH2H4folate), resulted in the appearance of a single protein band corresponding to the 1:1:1 enzyme-FdUMP-CH2H4folate complex, confirming the new species as dimeric thymidylate synthase containing a single functional active site.(ABSTRACT TRUNCATED AT 250 WORDS)
Bradshaw
T P
TP
Department of Chemistry and Biochemistry and School of Medicine, University of South Carolina, Columbia 29208.
Dunlap
R B
RB
D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008956:Models, Chemical; D011487:Protein Conformation; D013940:Thymidylate Synthase / Q000737:chemistry / Q000378:metabolism*
D004800:Enzymes, Immobilized; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase