The Active Site Cysteines of Thiolase

Inactivation of thiolase with iodo[1‐14C]acetamide has been shown to be due to its reaction with cysteine residues in the enzyme. [1‐14C]Carbamoylmethylcysteine was identified as the only product of the reaction and a study of the radioactive peptides that were isolated from a tryptic digest of the [1‐14C]carbamoylmethyl‐enzyme has shown that the reactive cysteines occur in a unique sequence extending to at least 26 amino acid residues in the primary structure.The enzyme‐substrate compound formed when thiolase reacts with [1‐14C]acetyl‐CoA is shown to contain [14C]acetyl groups bound in thioester linkage to at least three cysteines per mole. Moreover these residues of [14C]acetyl‐cysteine occur exclusively in a sequence that is identical to the sequence around the [14C]carbamoylmethylcysteine residues in the enzyme‐inhibitor compound, showing that the substrate and the inhibitior react with the same four cysteine residues in the enzyme.These results provide additional evidence for the chemical identity of the four subunits of thiolase.