Active rhodanese lacking nonessential sulfhydryl groups contains an unstable C-terminal domain and can be bound, inactivated, and reactivated by GroEL.
Mutation of all nonessential cysteine residues in rhodanese turns the enzyme into a form (C3S) that is fully active but less stable than wild type (WT). This less stable mutant allowed testing of two hypotheses; (a) the two domains of rhodanese are differentially stable, and (b) the chaperonin GroEL can bind better to less stable proteins. Reduced temperatures during expression and purification were required to limit inclusion bodies and obtain usable quantities of soluble C3S. C3S and WT have the same secondary structures by circular dichroism. C3S, in the absence of the substrate thiosulfate, is cleaved by trypsin to give a stable 21-kDa species. With thiosulfate, C3S is resistant to proteolysis. In contrast, wild type rhodanese is not proteolyzed significantly under any of the experimental conditions used here. Mass spectrometric analysis of bands from SDS gels of digested C3S indicated that the C-terminal domain of C3S was preferentially digested. Active C3S can exist in a state(s) recognized by GroEL, and it displays additional accessibility of tryptophans to acrylamide quenching. Unlike WT, the sulfur-loaded mutant form (C3S-ES) shows slow inactivation in the presence of GroEL. Both WT and C3S lacking transferred sulfur (WT-E and C3S-E) become inactivated. Inactivation is not due to irreversible covalent modification, since GroEL can reactivate both C3S-E and WT-E in the presence of GroES and ATP. C3S-E can be reactivated to 100%, the highest reactivation observed for any form of rhodanese. These results suggest that inactivation of C3S-E or WT-E is due to formation of an altered, labile conformation accessible from the native state. This conformation cannot as easily be achieved in the presence of the substrate, thiosulfate.
Ybarra
Jesse
J
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA. horowitz@biochem.uthscsa.edu
Bhattacharyya
Anusri Mitra
AM
Panda
Markandeswar
M
Horowitz
Paul M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018834:Chaperonin 60 / Q000378:metabolism*; D002850:Chromatography, Gel; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006868:Hydrolysis; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000378:metabolism*
D018834:Chaperonin 60; D017931:DNA Primers; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013884:Thiosulfate Sulfurtransferase