The effect of substrate analogs on the circular dichroic spectra of thymidylate synthetase from Lactobacillus casei.
Circular dichroism studies from 290 to 400 nm with the thymidylate synthetase from Lactobacillus casei revealed characteristic Cotton effects in the presence of various folate analogs plus 5-fluoro-2'-deoxyuridylate. Omission of either substrate analog prevented the appearance of the Cotton effects. When 5-fluoro-2'-deoxyuridylate and (+/-)-5,10-methylenetetrahydrofolate are mixed with the synthetase, a ternary complex results which yields distinctive minor negative ellipicity bands at 285 and 332 nm and a major negative ellipticity bands at 285 and 332 nm and a major positive band at 305 nm. Similar results were obtained with the ternary complex containing (+)-5,10-methylenetetrahydrofolate, but the enzymically inactive (-) diastereoisomer induced only the positive band at 305 nm. More intense Cotton effects were elicited by (+/-)-5,11-methylenetetrahydrohomofolate with a major positive ellipticity band at 308 nm and a minor negative band at 335 nm. A ternary complex was also formed with dihydrofolate, which provided a major circular dichroic band at 305 nm and a broad minor negative band in the region of 335 nm. Deoxyuridylate and thymidylate also formed ternary complexes with dihydrofolate, but their ellipicity bands were much less intense. Other folate analogs that formed ternary complexes with 5-fluoro-2'-deoxyuridylate to provide characteristic circular dichroic spectra were tetrahydrofolate, tetrahydrohomofolate, 10-methyltetrahydrofolate, and a 2-amino-4-hydroxyquinazoline derivative. By measuring the increment in ellipticity at 305 nm on addition of specific ligands to enzyme solutions, it was determined that the L. casei thymidylate synthetase contains two binding sites for 5-fluoro-2'-deoxyuridylate and for each of the diastereoisomers of 5,10-methylenetetrahydrofolate. An improved procedure is presented for the large-scale purification and crystallization of L. casei thymidylate synthetase.
D001665:Binding Sites; D002942:Circular Dichroism; D005468:Fluorodeoxyuridylate / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000494:pharmacology; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008780:Methyltransferases; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000378:metabolism; D013942:Thymine Nucleotides
D005493:Folic Acid Antagonists; D013942:Thymine Nucleotides; D005468:Fluorodeoxyuridylate; D008780:Methyltransferases; D013940:Thymidylate Synthase