title: The effects of an engineered cation site on the structure, activity, and EPR properties of cytochrome c peroxidase.
abstract: Earlier work [Bonagura et al. (1996) Biochemistry 35, 6107] showed that the K+ site found in the proximal pocket of ascorbate peroxidase (APX) could be engineered into cytochrome c peroxidase (CCP). Binding of K+ at the engineered site results in a loss in activity and destabilization of the CCP compound I Trp191 cationic radical owing to long-range electrostatic effects. The engineered CCP mutant crystal structure has been refined to 1.5 A using data obtained at cryogenic temperatures which provides a more detailed basis for comparison with the naturally occurring K+ site in APX. The characteristic EPR signal associated with the Trp191 radical becomes progressively weaker as K+ is added, which correlates well with the loss in enzyme activity as [K+] is increased. These results coupled with stopped-flow studies support our earlier conclusions that the loss in activity and EPR signal is due to destabilization of the Trp191 cationic radical.
authors.affiliation: Department of Molecular Biology & Biochemistry, University of California, Irvine 92697-3900, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Bonagura
authors.forename: M
authors.initials: M
authors.lastname: Sundaramoorthy
authors.forename: B
authors.initials: B
authors.lastname: Bhaskar
authors.forename: T L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002412:Cations / Q000138:chemical synthesis* / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D003578:Cytochrome-c Peroxidase / Q000138:chemical synthesis* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D011188:Potassium / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D002412:Cations; D011994:Recombinant Proteins; D003578:Cytochrome-c Peroxidase; D011188:Potassium