Interaction of deoxyuridylate with thymidylate synthetase.
The reaction of deoxyuridylate with Lactobacillus casei thymidylate synthetase (methylenetetrahydrofolate: deoxyuridylate C-methyltransferase) in the absence of folate or thiols can be followed by changes in the circular dichroic spectra at 267 nm. These changes at the maximum absorption wavelength of the pyrimidine suggest that on binding the interaction of the pyrimidine ring and the deoxyribose moiety is altered. The binding curve follows the form expected for single site binding. There was no change in the ultraviolet absorption spectra. The dissociation constant of the deoxyuridylate enzyme complex was calculated to be 4 x 10-minus 7 M. Iodoacetamide also reacts stoichiometrically with one of the two subunits of thymidylate synthetase and inactivates the enzyme. This reaction is blocked by the presence of deoxyuridylate or chloromercuribenzoate. Complement fixation and immunodiffusion studies gave no indication that iodoacetamide treatment caused conformational changes or breakdown of the enzyme into subunits. Treatment with 0.1 M mercaptoethanol restored activity to one-half the value obtained with native enzyme, suggesting that mercaptoethanol enabled the unreacted subunit to become functional.
D000596:Amino Acids / Q000032:analysis; D002250:Carbon Radioisotopes; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002942:Circular Dichroism; D003168:Complement Fixation Tests; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005779:Immunodiffusion; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008623:Mercaptoethanol / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000378:metabolism; D013940:Thymidylate Synthase / Q000276:immunology / Q000378:metabolism*; D014500:Uracil Nucleotides / Q000494:pharmacology*; D014542:Uridine Monophosphate / Q000494:pharmacology*
D000596:Amino Acids; D002250:Carbon Radioisotopes; D002729:Chloromercuribenzoates; D003854:Deoxyribonucleotides; D013763:Tetrahydrofolates; D014500:Uracil Nucleotides; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; D014542:Uridine Monophosphate; D008780:Methyltransferases; D013940:Thymidylate Synthase; D007460:Iodoacetamide