Active site cysteinyl and arginyl residues of rhodanese. A novel formation of disulfide bonds in the active site promoted by phenylglyoxal.

Chemical modification studies of bovine liver rhodanese have underscored important distinctions between free rhodanese and the catalytic intermediate in which the sulfane atom of the sulfur donor is bound covalently to the enzyme (sulfur-rhodanese). Treatment of free rhodanese with near-stoichiometric quantities of either iodoacetate or phenylglyoxal results in the rapid modification of the essential sulfhydryl group of Cys-247 and the consequent inactivation of the enzyme. Analysis of rate data for the iodoacetate reaction showed that the apparent pK of this group is 7.8 in free rhodanese and 6.7 to 7.0 in complexes of the enzyme with analogs of sulfur donor substrates, in agreement with the previous inference from steady state kinetic observations. Inactivation of free rhodanese by phenylglyoxal in the presence of cyanide was shown to be caused by a novel reaction in which disulfide bonds are formed between Cys-247 and either Cys-254 or Cys-263. In contrast to these results with free rhodanese, the sulfur-substituted enzyme is not inactivated by iodoacetate and is only relatively slowly inactivted by treatment with substantial excesses of phenylglyoxal. The loss of enzyme activity in sulfur-rhodanese does not involve cysteinyl residues but can be correlated with the modification of guanidino groups, notably that of Arg-186, the side chain of which may play a role in substrate binding. These chemical modification studies have implications with respect to the chemical mechanism of rhodanese catalysis and the interpretation of the x-ray crystallographic analysis of this enzyme.