Mutation of cysteine 254 facilitates the conformational changes accompanying the interconversion of persulfide-substituted and persulfide-free rhodanese.
Mutants of rhodanese (EC 2.8.1.1) that substitute Ser for Cys-254 and/or Cys-263, i.e. C254S, C263S, and the double mutant C254/263S, were compared with the wild-type enzyme to test the hypothesis that the persulfide formation to give sulfur-substituted rhodanese (ES) during catalysis can exert a stabilizing influence on the global structure of the protein. All the ES forms were quite refractory toward limited tryptic digestion. Extensive digestion was observed only in the persulfide-free forms (E) of mutants having substitutions of Ser for Cys-254 (C254/263S and C254S). Limited proteolysis of C254/263S produced a species on SDS gels at approximately 21 kDa that was proteolyzed further to give a slightly smaller product. Amino acid sequencing, together with mass spectrometry, indicated that these two components had the same N-terminal sequence as wild-type rhodanese and that they resulted from cleavage in the C-terminal domain, first at residues Arg-186 or Arg-182 and subsequently at Arg-175 or Lys-174. In addition to increased proteolytic susceptibility, the conversion from ES to E of C254/263S was accompanied by increased hydrophobic exposure and increased quenching of the intrinsic protein fluorescence by acrylamide. The substitution of Ser for Cys-254, within the helix suggested to be involved in stabilizing the persulfide bond in ES, seems to promote the formation of flexible conformations of sulfur-free rhodanese. In keeping with this model, C254/263S shows a marked increase in the hysteresis that is observed in rhodanese catalysis at pH 7 and high ionic strength. These results can be understood in terms of a model, suggesting that there is mutual stabilization of the protein structure and the persulfide bond in the ES form of rhodanese.
Islam
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TA
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760.
Miller-Martini
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DM
Horowitz
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PM
D000595:Amino Acid Sequence; D003545:Cysteine; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013440:Sulfides / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin
D011994:Recombinant Proteins; D013440:Sulfides; C051552:persulfides; D013884:Thiosulfate Sulfurtransferase; D014357:Trypsin; D003545:Cysteine