A comparison of substrate dynamics in human CYP2E1 and CYP2A6

• Published in: Biochemical and biophysical research communications Vol. 352; no. 4; pp. 843 - 849
• Main Authors: Harrelson, John P., Henne, Kirk R., Alonso, Darwin O.V., Nelson, Sidney D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26-01-2007
• Subjects: Allosterism; Aryl Hydrocarbon Hydroxylases - metabolism; Catalysis; Cooperativity; CYP2A6; CYP2E1; Cytochrome P-450 CYP2A6; Cytochrome P-450 CYP2E1 - metabolism; DEUTERIUM; Dynamics; ENZYMES; Humans; INHIBITION; Isotope effect; ISOTOPE EFFECTS; kH/ kD; Mixed Function Oxygenases - metabolism; Molecular Structure; OXIDATION; Oxidation-Reduction; P450; RADIATION, THERMAL, AND OTHER ENVIRONMENTAL POLLUTANT EFFECTS ON LIVING ORGANISMS AND BIOLOGICAL MATERIALS; Substrate Specificity; SUBSTRATES; Xylene; XYLENES; Xylenes - metabolism; CYP2A6; Isotope effect; k H/ k D; Dynamics; Allosterism; Xylene; P450; CYP2E1; Cooperativity
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2006.11.071

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.