Structural studies of bovine liver thiosulfate sulfurtransferase II. S-carboxymethylation of the enzyme and sequence analysis of the tryptic cysteinyl peptides

1. 1. Alkylation7 of the two free sulfhydryl groups in the monomer of thiosulfatesulfurtransferase A by iodo[¹⁴C]acetate has been studied with both the native enzyme and the reduced and denatured protein. Carboxymethylation of thiosulfate sulfurtransferase at 0 °C in 0.1 M glycine, pH 9.0, is attended by rapid inactivation of the enzyme and the structural modification is restricted essentially to one of the two cysteine residues. Inactivation of the enzyme by iodoacetate is also observed in a system in which glycine is replaced by 0.1 M Tris, pH 9.0, but under these conditions both cysteines undergo alkylation. The specific radioactivities of the tryptic [¹⁴C]carboxymethylcysteinyl peptides isolated from this derivative after reduction and alkylation with non-labeled reagent indicated that complete substitution of the active-site cysteine and 50% labeling of the second, non-essential sulfhydryl group had occurred in the Tris-buffer system. Modification of side chains other than the two sulfhydryl groups was not observed. 2. 2. Two tryptic [¹⁴C]carboxymethylcysteinyl peptides were isolated in high yield by gel filtration, ion-exchange chromatography and paper electrophoresis. One of the fragments, a basic dodecapeptide, was shown to contain the active-site cysteine; the amino acid sequence is Lys-Val-Asn-Leu-Thr-Cys(Cm)-Pro-Leu-Thr-Ile-Ala-Arg. The sequence of the second acidic octapeptide containing the non-essential cysteine is Thr-Cys(Cm)-Ser-Glu-Asp-Leu-Val-Arg. 3. 3. Studies of the effect of glycine on the alkylation of native thiosulfate sulfurtransferase suggest that this amino acid may block reaction with the two non-essential cysteine residues in enzyme dimer. The maximal labeling of these sites observed in the reaction of native dimer with iodo[¹⁴C]acetate in the absence of glycine was 50%. These findings provide support for the contention that the two non-essential sulfhydryl groups in thiosulfate sulfurtransferase dimer may be proximally located in a region of the molecule that binds glycine, and that alkylation of one sulfhydryl prevents reaction with its counterpart in the adjacent monomeric subunit.