Conversion of an Engineered Potassium-binding Site into a Calcium-selective Site in Cytochrome c Peroxidase
We have previously shown that the K + site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A., Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107â6115; B...
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Veröffentlicht in: | The Journal of biological chemistry 1999-12, Vol.274 (53), p.37827-37833 |
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Zusammenfassung: | We have previously shown that the K + site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A., Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107â6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538â5545). All other peroxidases bind Ca 2+ rather than K + . Using the K + -binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed
that should bind Ca 2+ selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na + , is bound instead of Ca 2+ . This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the
essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed
Ca 2+ binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands.
Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp 191 radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding
selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca 2+ more effectively than K + , demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.274.53.37827 |