An additional serine residue at the C terminus of rhodanese destabilizes the enzyme

Gisela Kramer, Vasanthi Ramachandiran, Paul Horowitz, Boyd Hardesty

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


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°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.

Original languageEnglish (US)
Pages (from-to)332-337
Number of pages6
JournalArchives of Biochemistry and Biophysics
Issue number2
StatePublished - Jan 15 2001
Externally publishedYes


  • In vitro coupled transcription/translation
  • Instability of variant rhodanese
  • Rhodanese
  • Rhodanese 3′ mutants

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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