NDLI: Cooperativity in oxidation reactions catalyzed by cytochrome P450 1A2: highly cooperative pyrene hydroxylation and multiphasic kinetics of ligand binding.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Stec, Donald F. Marsch, Glenn A. Isin, Emre M. Guengerich, F. Peter Sohl, Christal D. Eoff, Robert L.
Description	Author Affiliation: Sohl CD ( Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.)
Abstract	Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of alpha-naphthoflavone (alphaNF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for alphaNF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (alphaNF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-alphaNF complex show active site space for only one alphaNF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.
ISSN	00219258
e-ISSN	1083351X
Journal	Journal of Biological Chemistry
Issue Number	11
Volume Number	283
Language	English
Publisher	American Society for Biochemistry and Molecular Biology (United States)
Publisher Date	2008-03-14
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Cytochrome P-450 CYP1A2 Chemistry Oxygen Pyrenes Animals Metabolism Dose-Response Relationship, Drug Kinetics Ligands Magnetic Resonance Spectroscopy Models, Biological Models, Chemical Protein Binding Rabbits Spectrometry, Fluorescence Substrate Specificity Research Support, N.I.H., Extramural Biochemistry Molecular Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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