An additional serine residue at the C terminus of rhodanese destabilizes the enzyme.
The rhodanese coding sequence was extended at its 3' end by three base pairs to generate mutants coding for a serine or arginine residue at the carboxyl terminus of the protein. Wild-type and mutant coding sequences were expressed in a cell-free Escherichia coli system by coupled transcription/translation. Predominantly full-length protein was formed in all cases. The amount of protein synthesized was quantified by incorporation of radioactive leucine into polypeptides. Enzymatic activity of in vitro synthesized rhodanese was determined at different temperatures. Specific enzymatic activity was calculated and is assumed to reflect the portion of the protein that is in its native three-dimensional conformation. It was observed that rhodanese extended by one serine at the C terminus lost enzymatic activity when incubated above 30 degrees C, in contrast to wild-type protein or variant rhodanese extended by an arginine residue. Similarly, variant rhodanese with an additional serine residue was more susceptible to urea denaturation than the other two rhodanese species. These results are surprising in light of the crystal structure of the protein.
Kramer
G
G
Department of Chemistry and Biochemistry, University of Texas at Austin, 78712, USA. g.kramer@mail.utexas.edu
Ramachandiran
V
V
Horowitz
P
P
Hardesty
B
B
D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002474:Cell-Free System; D018835:Chaperonin 10 / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism; D003062:Codon / Q000235:genetics; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013884:Thiosulfate Sulfurtransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013885:Thiosulfates / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D018835:Chaperonin 10; D018834:Chaperonin 60; D003062:Codon; D013885:Thiosulfates; D012965:Sodium Chloride; D012694:Serine; D014508:Urea; D001120:Arginine; D013884:Thiosulfate Sulfurtransferase; D007930:Leucine