Cysteine 254 can cooperate with active site cysteine 247 in reactivation of 5,5'-dithiobis(2-nitrobenzoic acid)-inactivated rhodanese as determined by site-directed mutagenesis.
Sulfhydryl substitution mutants of rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) were used to determine whether the 4 cysteine residues in the native structure could cooperate in reactions. The sulfhydryl reactivity of persulfide-containing (ES) rhodanese was not significantly changed when cysteine residues at positions 63, 254, and 263 were replaced by serine, either individually or in combination. However, the sulfhydryl reactivity of persulfide-free (E) rhodanese was enhanced when Cys-254 was mutated. One sulfhydryl group, presumably the active site Cys-247, reacted rapidly in the E forms of these proteins with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 4,4'-dipyridyl disulfide (4-PDS). After reaction with DTNB or 4-PDS, proteins with Cys-254 retained > 95% of their original activities as compared with Ser-254-containing proteins, which retained < 6% of their activities. Cyanide treatment could release thionitrobenzoate from rhodanese-thionitrobenzoate complexes with an approximate 1:1 stoichiometry. After this treatment, only the wild-type and C263S enzymes were fully active. Cyanide-treated rhodanese-thionitrobenzoate complexes of the C254S and C254S/C263S mutants could be fully reactivated using an exogenously added thiol, beta-mercaptoethanol. These results are consistent with the formation of a Cys-247-thiocyano derivative that is inactive but capable of being reactivated by intramolecular transfer of cyanide to Cys-254. In the absence of Cys-254, beta-mercaptoethanol can serve as the transferring sulfhydryl group.
Miller-Martini
D M
DM
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Hua
S
S
Horowitz
P M
PM
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011725:Pyridines / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013440:Sulfides / Q000032:analysis; D013884:Thiosulfate Sulfurtransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D004793:Enzyme Reactivators; D011725:Pyridines; D013439:Sulfhydryl Reagents; D013440:Sulfides; C007542:4,4'-dipyridyl disulfide; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; D013884:Thiosulfate Sulfurtransferase; D003545:Cysteine