A comparison of substrate dynamics in human CYP2E1 and CYP2A6.
Considering the dynamic nature of CYPs, methods that reveal information about substrate and enzyme dynamics are necessary to generate predictive models. To compare substrate dynamics in CYP2E1 and CYP2A6, intramolecular isotope effect experiments were conducted, using deuterium labeled substrates: o-xylene, m-xylene, p-xylene, 2,6-dimethylnaphthalene, and 4,4'-dimethylbiphenyl. Competitive intermolecular experiments were also conducted using d(0)- and d(6)-labeled p-xylene. Both CYP2E1 and CYP2A6 displayed full isotope effect expression for o-xylene oxidation and almost complete suppression for dimethylbiphenyl. Interestingly, (k(H)/k(D))(obs) for d(3)-p-xylene oxidation ((k(H)/k(D))(obs)=6.04 and (k(H)/k(D))(obs)=5.53 for CYP2E1 and CYP2A6, respectively) was only slightly higher than (k(H)/k(D))(obs) for d(3)-dimethylnaphthalene ((k(H)/k(D))(obs)=5.50 and (k(H)/k(D))(obs)=4.96, respectively). One explanation is that in some instances (k(H)/k(D))(obs) values are generated by the presence of two substrates-bound simultaneously to the CYP. Speculatively, if this explanation is valid, then intramolecular isotope effect experiments should be useful in the mechanistic investigation of P450 cooperativity.
Harrelson
John P
JP
Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195, USA. harrelsonj@pacificu.edu
Henne
Kirk R
KR
Alonso
Darwin O V
DO
Nelson
Sidney D
SD
D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002384:Catalysis; D065637:Cytochrome P-450 CYP2A6; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism*; D006801:Humans; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D014992:Xylenes / Q000378:metabolism
D014992:Xylenes; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; C031285:3-xylene