Related Experiment Video
Updated: Nov 18, 2025
Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling
Published on: March 20, 2018
Dynamics underlying hydroxylation selectivity of cytochrome P450cam
Sashary Ramos1, Claire C Mammoser1, Katherine E Thibodeau1
1Department of Chemistry, Indiana University, Bloomington, Indiana.
Site-specific two-dimensional infrared (IR) spectroscopy reveals how enzyme structural dynamics influence substrate selectivity. This method elucidates the mechanism behind cytochrome P450
Area of Science:
- Biochemistry
- Spectroscopy
- Enzymology
Background:
- Enzyme catalysis selectivity is governed by complex structural dynamics and heterogeneity.
- Characterizing these dynamics at the amino acid level and rapid timescales remains a significant challenge.
Purpose of the Study:
- To investigate the mechanism of lower regioselectivity in cytochrome P450 (P450cam) hydroxylation of norcamphor compared to camphor.
- To demonstrate the utility of site-specific two-dimensional infrared (IR) spectroscopy for probing enzyme-substrate interactions.
Main Methods:
- Utilized site-specific two-dimensional IR spectroscopy with cyano group vibrational probes.
- Applied linear and 2D IR spectroscopy to measure heterogeneity and dynamics at targeted enzyme locations.
- Compared P450cam complexes with camphor and norcamphor substrates.
Main Results:
- Norcamphor complexation does not induce the same large-scale conformational closure as camphor.
- Probes on the bound norcamphor substrate reveal rapidly interconverting states.
- These dynamics correlate with the observed hydroxylation product distribution.
Conclusions:
- Large- and small-scale structural heterogeneity and dynamics significantly contribute to cytochrome P450 selectivity.
- Site-selective IR spectroscopy is a powerful approach for elucidating protein dynamics and enzyme mechanisms.
More Related Videos
08:02Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory
Published on: August 23, 2018
09:42Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
Published on: January 16, 2016
Related Concept Videos
01:17Drug Metabolism: Phase I Reactions
02:36Regioselectivity and Stereochemistry of Hydroboration
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn stereochemistry.
02:17Reduction of Alkenes: Asymmetric Catalytic Hydrogenation
The metal catalyst used can be either heterogeneous or homogeneous. When hydrogenation of an alkene generates a chiral center, a pair of enantiomeric products is expected to form. However, an enantiomeric excess of one of the products can be facilitated using an enantioselective reaction or an...
02:17Regioselectivity of Electrophilic Additions to Alkenes: Markovnikov's Rule
The hydrohalogenation of an unsymmetrical alkene can yield two haloalkane products, depending on which vinylic carbon takes up the halogen. However, one product usually predominates, where hydrogen adds to the vinylic carbon bearing the...
02:13Reduction of Alkenes: Catalytic Hydrogenation
Metals like palladium, platinum, and nickel are commonly used in their solid forms — fine powder on an inert surface. As these catalysts remain insoluble in the reaction mixture, they are referred to as heterogeneous catalysts.
The hydrogenation process takes place on the...
02:34Regioselectivity and Stereochemistry of Acid-Catalyzed Hydration