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 pair s to generate mutants coding for a serine or arginine residue at the carbox yl terminus of the protein. Wild-type and mutant coding sequences were expr essed in a cell-free Escherichia coil system by coupled transcription/trans lation. Predominantly full-length protein was formed in all eases. The amou nt of protein synthesized was quantified by incorporation of radioactive le ucine into polypeptides, Enzymatic activity of in vitro synthesized rhodane se was determined at different temperatures. Specific enzymatic activity wa s calculated and is assumed to reflect the portion of the protein that is i n its native three-dimensional conformation. It was observed that rhodanese extended by one serine at the C terminus lost enzymatic activity when incu bated above 30 degreesC, in contrast to wild-type protein or variant rhodan ese extended by an arginine residue. Similarly variant rhodanese with an ad ditional serine residue was more susceptible to urea denaturation than the other two rhodanese species. These results are surprising in light of the c rystal structure of the protein, (C) 2001 Academic Press.