title: Identification and reactivity of the catalytic site of pig liver thioltransferase.
abstract: The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D003553:Cystine / Q000378:metabolism; D054477:Glutaredoxins; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D011490:Protein Disulfide Reductase (Glutathione); D013552:Swine
chemical_list: D002250:Carbon Radioisotopes; D054477:Glutaredoxins; D007461:Iodoacetates; D003553:Cystine; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D019807:Iodoacetic Acid