When yeast fatty acid synthetase is treated with iodoacetamide at 0?C, three carbamoylmethyl residues are incorporated into the protein under concomitant complete loss of the enzymatic activity [Oesterhelt, D., Bauer, H., Kresze, G.-B., Steber, L. and Lynen, F. (1977) Eur. J. Biochem. 79, 173?180]. Here we report that, in this reaction, only cysteinyl residues are alkylated. During protein hydrolysis of the modified enzyme, S-carboxymethyl cysteine is formed which, when treated with performic acid in the presence of a strong acid and chloride ions, is oxidized only partially to yield open-chain S-carboxymethyl cysteine sulfone but, for the greater part, is transformed into S-carboxymethyl cysteine lactam sulfone (3-carboxy-5-oxo-tetrahydro-1,4-thiazine-1,1-dioxide), a novel derivative of S-carboxymethyl cysteine which cannot be detected with ninhydrin. If, on the other hand, the carbamoylmethylated enzyme is treated first with performic acid and hydrolyzed afterwards, a large part of the carbamoylmethyl residues is lost due to decarboxylation of the α-sulfonyl carboxylic acid S-carboxymethyl cysteine sulfone. When carboxamidomethylated fatty acid synthetase was digested with trypsin at 0?C, two different carbamoylmethyl peptides were found which could be separated by gel filtration. The larger peptide TA was cleaved, during prolongated incubation with trypsin at 30?C, to yield the smaller peptide TB which was purified and its amino acid sequence determined to be Thr-Pro-Val-Gly-Ala-Cys(carbamoylmethyl). By comparison of tryptic and peptic peptides from acetylated and carboxamidomethylated synthetase, respectively, it is demonstrated that the same cysteinyl residues are alkylated by iodoacetamide which are the acetyl-binding sites of the condensing enzyme component, i.e. the peripheral SH-groups.

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Kresze GB, Steber L, Oesterhelt D, Lynen F

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