title: Examining reactivity and specificity of cytochrome c peroxidase by using combinatorial mutagenesis.
abstract: Combinatorial mutagenesis was used to investigate the role of three key residues in cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae, Arg48, Trp51, and Trp191, in control of the reactivity and selectivity of the heme-containing enzyme. Libraries were prepared by randomization of these residues and were subsequently screened for activity against the phenolic substrate guaiacol. Screening conditions were employed that favor either mutants with high activity or those with both high activity and stability of the reactive enzyme intermediates. The results obtained suggest a dual role for Arg48 of CCP: in addition to stabilizing reactive enzyme intermediates, the distal arginine residue plays a major role in restriction of access to the ferryl oxygen atom by small molecules and thereby controls reactivity and substrate specificity of the peroxidase. At position 51 of CCP, either a phenylalanine or a tryptophan residue is required both for catalytic and structural reasons. In contrast, either polar or positively charged residues are accepted at the position of Trp191, which is located inside the core of the protein. The variability at position 191 can be interpreted as a reflection of the mechanism of cytochrome c peroxidase, which transforms the nonpolar Trp191 into a transient cation radical.
authors.affiliation: Swiss Federal Institute of Technology Lausanne (EPFL) Institute of Molecular and Biological Chemistry 1015 Lausanne, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Wilming
authors.forename: André
authors.initials: A
authors.lastname: Iffland
authors.forename: Petra
authors.initials: P
authors.lastname: Tafelmeyer
authors.forename: Claudio
authors.initials: C
authors.lastname: Arrivoli
authors.forename: Christophe
authors.initials: C
authors.lastname: Saudan
authors.forename: Kai
authors.initials: K
authors.lastname: Johnsson
mesh_terms: D019943:Amino Acid Substitution; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D003578:Cytochrome-c Peroxidase