Interpretations of cytochrome P450 mechanisms from kinetic studies.
The catalytic mechanism of cytochrome P450 (P450) enzymes has generally been understood in terms of a classic cycle in which electron donation is often limiting and catalysis is understood in terms of hydrogen abstraction and rapid oxygen rebound. In the course of detailed investigations with kinetic hydrogen isotope effects we have studied two systems in which somewhat unusual isotope effects have been interpreted in terms of modifications of the general paradigm. The low isotope effects observed for N-demethylation reactions are in contrast to high values seen with P450-catalyzed C-hydroxylation and peroxidase-catalyzed N-demethylation and are consonant with a role for the P450 FeO2+ entity in base-catalyzed deprotonation of an aminium radical. With P450 2E1, kinetic deuterium isotope effects are seen on the apparent Km for the substrate (increased) but not on Vmax. The results are interpreted in terms of a mechanism where C-H bond cleavage is sensitive to deuterium substitution but a step following this is rate-limiting. This step may be product release.
Guengerich
F P
FP
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Bell
L C
LC
Okazaki
O
O
D000818:Animals; D002384:Catalysis; D002412:Cations; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003627:Data Interpretation, Statistical; D003640:Dealkylation; D003903:Deuterium; D005296:Ferrous Compounds / Q000737:chemistry*; D005609:Free Radicals; D006420:Hemeproteins / Q000378:metabolism; D006706:Homeostasis; D007700:Kinetics; D011522:Protons; D051381:Rats; D014316:Tritium
D002412:Cations; D005296:Ferrous Compounds; D005609:Free Radicals; D006420:Hemeproteins; D011522:Protons; D014316:Tritium; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; C034236:ferrous oxide