The effect of raney nickel on the covalent thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate complex

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Abstract
Raney Ni [Ni(H)] catalyzes a specific reductive cleavage of C-S bonds and 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 was denatured and then shaken with Ni(H) at 25.degree. 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 casel grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both 3H and 35S at identical rates. Control experiments on amino acids showed that only the S-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 ternary 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 RNase, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated RNase 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.