The subunit structure of rabbit skeletal muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group

The single highly reactive (class I) thiol group per 80,000 mol.wt. subunit of skeletal muscle phosphofructokinase was specifically carboxymethylated with iodo[2 ¹⁴C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2 ³H]acetate. After tryptic digestion and peptide 'mapping' it was found that the ¹⁴C radioactivity was in a spot that did not contain significant amounts of ³H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class I cysteine peptide. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80,000) being composed of two identical amino acid sequences. The amino acid sequence of the tryptic peptide containing the class I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.