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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.

The cationic site of the enzyme rhodanese forms ion pairs with the substrate and product anions, thiosulfate, cyanide, sulfite, and thiocyanate. It also forms ion pairs with such diverse anionic species as acetate, formate, sulfate, and glycinate. In rate studies, the equilibrium constants and their temperature dependencies were evaluated for the interactions of most of these anions with both the free enzyme and the sulfur-substituted enzyme intermediate. The ionic strength dependencies of the constants for the substrate, thiosulfate ion, and the inhibitor, sulfate ion, were also determined. The results of this study provide a basis for calculating the relative concentrations of all enzymic species under a broad variety of experimental conditions. Further, the study of the thermodynamic parameters indicates that the enzyme-anion interactions take place in an hydrophobic environment in which ion solvation is minimal. When the parameters of substrate and product origin are compared with those for the other enzyme-anion interactions, the results suggest that the protein undergoes conformational changes during the reaction. The direction of the entropy differences indicates that there are successive changes to a more constrained state on binding of the substrate, thiosulfate ion, and release of the product, sulfite ion, followed by a relaxation of the protein upon release of the product, thiocyanate ion.