title: Substrates modulate the rate-determining step for CO binding in cytochrome P450cam (CYP101). A high-pressure stopped-flow study.
abstract: The high-pressure stopped-flow technique is applied to study the CO binding in cytochrome P450cam (P450cam) bound with homologous substrates (1R-camphor, camphane, norcamphor and norbornane) and in the substrate-free protein. The activation volume DeltaV # of the CO on-rate is positive for P450cam bound with substrates that do not contain methyl groups. The kon rate constant for these substrate complexes is in the order of 3 x 10(6) M(-1) x s(-1). In contrast, P450cam complexed with substrates carrying methyl groups show a negative activation volume and a low kon rate constant of approximately 3 x 10(4) M(-1) x s(-1). By relating kon and DeltaV # with values for the compressibility and the influx rate of water for the heme pocket of the substrate complexes it is concluded that the positive activation volume is indicative for a loosely bound substrate that guarantees a high solvent accessibility for the heme pocket and a very compressible active site. In addition, subconformers have been found for the substrate-free and camphane-bound protein which show different CO binding kinetics.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, Protein Dynamics Laboratory, Berlin, Germany. cjung@mdc-berlin.de
authors.forename: Christiane
authors.initials: C
authors.lastname: Jung
authors.forename: Nicole
authors.initials: N
authors.lastname: Bec
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lange
mesh_terms: D001665:Binding Sites; D001892:Camphanes / Q000378:metabolism; D002164:Camphor / Q000378:metabolism*; D019475:Camphor 5-Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism*; D007700:Kinetics; D009636:Norbornanes / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001892:Camphanes; D009636:Norbornanes; C067796:camphane; C055059:norcamphor; D002164:Camphor; D002248:Carbon Monoxide; D019475:Camphor 5-Monooxygenase