Investigating the Structural Plasticity of a Cytochrome P450
| Autor: | Steven G. Kendrew, Per Jemth, Linda Celeste Montemiglio, Carmelinda Savino, Adriana E. Miele, Giuliano Sciara, Beatrice Vallone, Stefano Gianni |
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| Rok vydání: | 2009 |
| Předmět: |
0303 health sciences
biology Cytochrome Stereochemistry Chemistry 030302 biochemistry & molecular biology Cytochrome P450 Substrate (chemistry) Cell Biology biology.organism_classification Ligand (biochemistry) Biochemistry Receptor–ligand kinetics 03 medical and health sciences Structural plasticity biology.protein Saccharopolyspora erythraea Molecular Biology Drug metabolism 030304 developmental biology |
| Zdroj: | Journal of Biological Chemistry. 284:29170-29179 |
| ISSN: | 0021-9258 |
| Popis: | Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism. |
| Databáze: | OpenAIRE |
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