Functional analysis of human microsomal epoxide hydrolase genetic variants.
Human microsomal epoxide hydrolase (EPHX1) is active in the metabolism of many potentially carcinogenic or otherwise genotoxic epoxides, such as those derived from the oxidation of polyaromatic hydrocarbons. EPHX1 is polymorphic and encodes allelic variation at least two amino acid positions, Y113H and H139R. In a number of recent molecular epidemiological investigations, EPHX1 polymorphism has been suggested as a susceptibility factor for several human diseases. To better evaluate the functional contribution of EPHX1 genetic polymorphism, we characterized the enzymatic properties associated with each of the respective variant proteins. Enzymatic profiles were evaluated with cis-stilbene oxide (cSO) and benzo[a]pyrene-4,5-epoxide (BaPO), two prototypical substrates for the hydrolase. In one series of experiments, activities of recombinant EPHX1 proteins were analyzed subsequent to their expression using the pFastbac baculovirus vector in Spodoptera frugiperda-9 (Sf9) insect cells, and purification by column chromatography. In parallel studies, EPHX1 activities were evaluated with human liver microsomes derived from individuals of known EPHX1 genotype. Using the purified protein preparations, rates of cSO and BaPO hydrolysis for the reference protein, Y113/H139, were approximately 2-fold greater than those measured with the other EPHX1 allelic variants. However, when activities were analyzed using human liver microsomal fractions, no major differences were evident in the reaction rates generated among preparations representing the different EPHX1 alleles. Collectively, these results suggest that the structural differences encoded by the Y113H and H139R variant alleles exert only modest impact on EPHX1-specific enzymatic activities in vivo.
Hosagrahara
Vinayak P
VP
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105, USA.
Rettie
Allan E
AE
Hassett
Christopher
C
Omiecinski
Curtis J
CJ
D001483:Base Sequence; D017931:DNA Primers; D004851:Epoxide Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic
D017931:DNA Primers; D004851:Epoxide Hydrolases