Reaction of Yeast Fatty Acid Synthetase with Iodoacetamide 2. Identification of the Amino Acid Residues Reacting with Iodoacetamide and Primary Structure of a Peptide Containing the Peripheral Sulfhydryl Group.

When yeast fatty acid synthetase is treated with iodoacetamide at 0 °C, three carbamoyimethyl 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 T_A was cleaved, during prolongated incubation with trypsin at 30 °C, to yield the smaller peptide T_B 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. [ABSTRACT FROM AUTHOR]