Rhodanese conformational changes permit oxidation to give disulfides that form in a kinetically determined sequence.
When the structure of the monomeric enzyme rhodanese is perturbed by urea or SDS and heat, sulfhydryl assays combined with SDS gel analyses reveal that intrachain disulfides are formed rapidly. Two intrachain disulfide bonded species can be distinguished. One contains a single disulfide and comigrates on SDS gels with fully reduced rhodanese (Band I), while a second species contains two disulfides and migrates faster than the reduced enzyme (Band II). The kinetic path and identity of the participating sulfhydryl groups are suggested by the results with sulfhydryl mutants. On mild oxidation or perturbation, a single disulfide forms that involves two of the three sulfhydryl groups in C-terminal domain of the protein, i.e., two of the sulfhydryl groups from among the three residues: the active-site Cys-247, Cys-254 and Cys-263. These disulfides are the same as those that are formed upon oxidation of the native enzyme. The remaining sulfhydryl group of these three, in a kinetically slower process, can form a disulfide with Cys-63 which is in the N-terminal domain in native rhodanese. The resulting looped structure is so conformationally constrained that its shape and/or altered SDS binding gives rise to the 'fast' Band II on the SDS gels. The conformationally constrained species with two disulfides may be related to oxidized rhodanese species that are difficult to reduce.
Horowitz
P M
PM
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760, USA.
Hua
S
S
D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins; D006358:Hot Temperature; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry*; D014508:Urea
D004220:Disulfides; D005452:Fluoresceins; C015296:5-iodoacetamidofluorescein; D014508:Urea; D013884:Thiosulfate Sulfurtransferase; D003545:Cysteine