Studies on the Active Site of Rhodanese

The complete loss of enzymic activity on reaction with alkylating agents, aromatic nitro compounds, or aliphatic mercaptans indicates the importance of sulfhydryl groups for catalysis. Analyses during the course of inactivation with any of these reagents revealed the loss of one of the two —SH groups in the rhodanese monomer when inactivation was complete. Amino acid analysis of iodoacetate-inactivated enzyme showed that half its cysteine residues had been carboxymethylated. Sedimentation experiments showed that dinitrobenzene-inactivated enzyme was an oxidized dimer, indicating the formation of an intermolecular disulfide bond between monomers. Reduction of mercaptoethanol-inactivated enzyme resulted in the appearance of two —SH-groups, suggesting the existence of a mixed disulfide of the reagent and the enzyme. In addition, competitive inhibition of rhodanese activity by aromatic ions, but not by the corresponding aliphatics, supported previous evidence suggesting the presence of a tryptophanyl residue at the active center. Further results suggested close proximity of the essential sulfhydryl and aromatic groups in the active site of rhodanese.