The effect of Raney nickel on the covalent thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate complex.
Raney nickel (Ni(H)) catalyzes a specific reductive cleavage of carbon-sulfur bonds and, therefore, can be used to determine whether compounds are covalently bound to proteins through a sulfide linkage. When the covalent thymidylate synthetase-[3H]5-fluoro-2'-deoxyuridylic acid-[14C]-5,10-CH2H4-folate complex (Langenbach et al. (1972a), Biochem, Biophys. Res. Commun. 48, 1565) was denatured and then shaken with Ni(H) at 25 degrees C, both isotopes were rapidly cleaved from the protein, with identical reaction halftimes of less than 10 min. The liberated radioactivity was filterable through nitro-cellulose filters and comigrated with small molecules on Sephadex G-25. Both labels migrated identically upon paper chromatography. A [3H]5-fluoro-2'-deoxyuridylic acid-[35S]thymidylate synthetase complex was formed with enzyme isolated from Lactobacillus casei grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both tritium and sulfur-35 at identical rates. Control experiments on amino acids showed that only the sulfur-containing amino acids are degraded by Ni(H). Cysteine was rapidly converted to alanine and methionine to alpha-aminobutyric acid. 5-Carboxymethylcysteine and 5-uracilylcysteine, simple models for the tenary enzyme-5-fluoro-2'-deoxyuridylic acid-5,10-CH2H4-folate complex, were converted to alanine at the same rate that 5-fluoro-2'-deoxyuridylic acid (FdUrd-5'-P) was cleaved from the enzyme. Native ribonuclease, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated ribonuclease was desulfurized. Amino acid analysis of Ni(H)-treated thymidylate synthetase showed that cysteine was the only amino acid degraded. Gel electrophoresis of the proteins after exposure to Ni(H) showed no breakage of polypeptide chains. These results support a sulfide linkage between FdUrd-5'-P and thymidylate synthetase in the covalent complex.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D005468:Fluorodeoxyuridylate; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism; D009532:Nickel / Q000494:pharmacology; D011485:Protein Binding; D012260:Ribonucleases; D013438:Sulfhydryl Compounds; D013763:Tetrahydrofolates; D013940:Thymidylate Synthase / Q000378:metabolism; D014500:Uracil Nucleotides
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D013763:Tetrahydrofolates; D014500:Uracil Nucleotides; D005468:Fluorodeoxyuridylate; D009532:Nickel; D008780:Methyltransferases; D013940:Thymidylate Synthase; D012260:Ribonucleases