Examining reactivity and specificity of cytochrome c peroxidase by using combinatorial mutagenesis.

Details

Serval ID
serval:BIB_2151B6EA378B
Type
Article: article from journal or magazin.
Collection
Publications
Title
Examining reactivity and specificity of cytochrome c peroxidase by using combinatorial mutagenesis.
Journal
Chembiochem
Author(s)
Wilming M., Iffland A., Tafelmeyer P., Arrivoli C., Saudan C., Johnsson K.
ISSN
1439-4227
Publication state
Published
Issued date
2002
Peer-reviewed
Oui
Volume
3
Number
11
Pages
1097-1104
Language
english
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.
Keywords
Amino Acid Substitution, Cytochrome-c Peroxidase/chemistry, Cytochrome-c Peroxidase/metabolism, Directed Molecular Evolution, Mutagenesis, Saccharomyces cerevisiae/enzymology
Pubmed
Web of science
Create date
19/05/2008 11:00
Last modification date
20/08/2019 12:57
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