SITE-DIRECTED REPLACEMENT OF THE INVARIANT LYSINE 73 OF SACCHAROMYCES CEREVISIAE ISO-1-CYTOCHROME C WITH ALL RIBOSOMALLY ENCODED AMINO ACIDS

摘要

Site-directed mutagenesis has been used to replace the evolutionally invariant lysine 73 of Saccharomyces cerevisiae iso-1-cytochrome c with all 19 ribosomally encoded amino acids. This residue is implicated in the protein-protein complexation and redox properties of cytochrome c. Functional studies were performed in the context of both a multicopy and a centromeric phagemid using a yeast strain that is incapable of iso-1-cytochrome c production. Functionality was ascertained by growth on the non-fermentable carbon sources lactate and glycerol at 30 degrees C and 37 degrees C. Doubling times derived from growth curves in liquid glycerol media were used to quantify differences in the functionality of mutants relative to the wild type protein. The cysteine 73 mutant shows a dramatically reduced growth rate. Growth rates for transformants carrying the other mutations are no more than 50% slower than transformants carrying the wild type allele. The data suggest that thermostability is related to evolutionary conservation but that the requirement for a lysine residue at position 73 is met readily by a variety of amino acids. Therefore, the redox properties of cytochrome c and its complexation with its redox partners are not absolutely dependent on a lysine at position 73. [References: 41]