title: Effect of alcohols on binding of camphor to cytochrome P450cam: spectroscopic and stopped flow transient kinetic studies.
abstract: Addition of alcohols to cytochrome P450cam (CYP101) was shown to release the substrate camphor from the heme pocket of the enzyme. The release of the substrate was found to be caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme. The far-UV CD and near-UV CD spectra reveal that addition of alcohols to cytochrome P450cam cause a small change in the secondary structural elements but a significant change in the tertiary structural organization of this enzyme. The CD spectra at the heme region at various concentrations of alcohols indicate a substantial change in the tertiary structural organization around the heme moiety too. The equilibrium constant associated with the binding of camphor to Cyt P450cam is strongly dependent on the concentration of alcohols and the corresponding free energy associated with the binding is found to scale linearly with the concentration of alcohols. Kinetic experiments on binding of camphor to Cyt P450cam show that both k(on) and k(off) rate constants are strongly affected by addition of alcohols suggesting that alcohol expel camphor out of the heme cavity of Cyt P450cam by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
authors.forename: R
authors.initials: R
authors.lastname: Murugan
authors.forename: Shyamalava
authors.initials: S
authors.lastname: Mazumdar
mesh_terms: D000438:Alcohols / Q000737:chemistry* / Q000494:pharmacology*; D001665:Binding Sites; D002164:Camphor / Q000737:chemistry*; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D004789:Enzyme Activation; D017022:Flow Injection Analysis; D007700:Kinetics; D011485:Protein Binding; D013057:Spectrum Analysis; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D002164:Camphor; D019475:Camphor 5-Monooxygenase