6533b85efe1ef96bd12bf372

RESEARCH PRODUCT

Sequestration of biological reactive intermediates by trapping as covalent enzyme-intermediate complex

subject

description

One important class of biological reactive intermediates arising in the course of human xenobiotic metabolism are arene and alkene oxides. The major safeguard against the potential genotoxic effects of these compounds is the microsomal epoxide hydrolase (mEH). This enzyme has a broad substrate specificity but--on the first sight--seems to be inadequately suited for this protection task due to its low turnover number with most of its substrates. The recent progress in the understanding of the mechanism of enzymatic epoxide hydrolysis has shed new light on this apparent dilemma: Epoxide hydrolases convert their substrates via the intermediate formation of a covalent enzyme-substrate complex, and it has been shown that the formation of the intermediate proceeds by orders of magnitudes faster than the subsequent hydrolysis, i.e. the formation of the terminal product. Thus, the enzyme acts like a molecular sponge by binding and inactivating the dangerous metabolite very fast while the subsequent product release is considerably slower, and quantification of the latter heavily underestimates the speed of detoxification. Usually, the slow enzyme regeneration does not pose a problem, since the mEH is highly abundant in human liver, the organ with the highest capacity to metabolically generate epoxides. Computer simulation provides evidence that the high amount of mEH enzyme is crucial for the control of the steady-state level of a substrate epoxide and can keep it extremely low. Once the mEH is titrated out under conditions of extraordinarily high epoxide concentration, the epoxide steady-state level steeply rises, leading to a sudden burst of the genotoxic effect. This prediction of the computer simulation is in perfect agreement with our experimental work. V79 Chinese Hamster cells that we have genetically engineered to express human mEH at about the same level as that observed in human liver are well protected from any measurable genotoxic effect of the model compound styrene oxide (STO) up to an apparent threshold level of 100 microM in the cell culture medium. In V79 cells that do not express mEH, STO triggers the formation of DNA strand breaks in a dose-dependent manner with no apparent threshold. Above 100 microM, the genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line.