The sulfurtransferase activity and structure of rhodanese are affected by site-directed replacement of Arg-186 or Lys-249.
Two mutants of the enzyme rhodanese that replace Arg-186 with Leu (R186L) or Lys-249 with Ala (K249A) were prepared to test suggestions that these residues are involved in catalysis and structure. The predominant effect with R186L was functional, and Km for the sulfur donor, thiosulfate, increased from 3.7 mM to 73 mM with a modest decrease in Vmax (672 IU/mg to 576 IU/mg). However, K249A was virtually inactive using thiosulfate, but it was active with thiosulfonates such as p-toluene-, 2-aminoethane-, or ethanethiosulfonate, and these compounds could be demonstrated to form persulfide-substituted rhodanese. Compared with wild type enzyme, K249A had (a) reduced stability, (b) comparable secondary structure, (c) more easily exposed hydrophobic surfaces, and (d) a core structure that denatured similarly to the wild type enzyme. Thus, Arg-186 and Lys-249 are important in rhodanese catalysis, and Lys-249 is particularly critical for substrate selectivity and protein stability. Finally, the results suggest that there can be active rhodanese species in vivo that will be undetected using thiosulfate as a sulfur donor.
Luo
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GX
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Horowitz
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PM
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014508:Urea; D001120:Arginine; D013884:Thiosulfate Sulfurtransferase; D008239:Lysine