CYP2E1 Substrate Inhibition

Abstract

In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min-1) with relatively low Km (24 μm); nevertheless, at concentrations of >100 μm, the rate of pNP oxidation gradually decreased as a second at the was a for a inhibition for the was we the inhibition a the of with a for the for pNP a the the of the of the of the the the of for the the of at the at the In this study we offer a mechanistic interpretation of the previously known but unexplained substrate inhibition observed for CYP2E1. At low substrate concentrations, p-nitrophenol (pNP) was rapidly turned over (47 min-1) with relatively low Km (24 μm); nevertheless, at concentrations of >100 μm, the rate of pNP oxidation gradually decreased as a second at the was a for a inhibition for the was we the inhibition a the of with a for the for pNP a the the of the of the of the the the of for the the of at the at the for a a a of of the of of for as the the of of the of as of with the for the for for of as substrate the this a of the p-nitrophenol a of CYP2E1. the a pNP for a of for the a a the of substrate inhibition for pNP of the the pNP a the observed rate of a was the rate gradually decreased at pNP the was a substrate inhibition the the substrate inhibition of a second with the substrate concentrations this decreased for the of for was inhibition with of a of pNP the of a for the the for was of was the of concentrations of pNP for the of we this of mechanistic of we as for oxidation of the of inhibition In as the of the a of the CYP2E1. pNP was for of the pNP the the of we for the the the a of a we the of of with a of a second for a the the of this study the for was was of the In In with was as a pNP for oxidation of pNP a was with a pNP was the the of of with a was the concentrations of a second for of for the the of the for concentrations for this of at for of a was at a with was a low a the for the previously of a with a at a rate of of the pNP at the was concentrations as a of the rate was with the of the observed substrate inhibition pNP oxidation for for substrate nevertheless, of inhibition the for the at the the substrate substrate was the of for In substrate of the substrate substrate but substrate the was the In this the substrate substrate In the of inhibition the the inhibition for pNP the for inhibition the of as the for this the of for of the of the of a for In this we the the the low for the of the of the the decreased the as a of for the of with for observed we with of at was the a In the we the with the of the with for with the the of a the observed substrate inhibition for pNP oxidation the of was the of with we the as for of the we we the a as the for this for of was the the of the a for the the of the the with the was of the the of CYP2E1. a the the a of the of substrate previously oxidation of pNP substrate substrate a a the for a substrate substrate we the a as a relatively low (24 the of for nevertheless, at pNP concentrations the gradually decreased as a second at the of was inhibition of of oxidation of pNP the of pNP at was as a of as the the for was the of at a of the for pNP a of for we the of inhibition of the observed substrate inhibition pNP oxidation for inhibition for substrate nevertheless, of inhibition substrate we a the the of the as a the for the the for for the was a for for In this pNP for the at the of the the concentrations of the for the second we a second of of the over with as for inhibition of pNP inhibition a of for inhibition of pNP of a the for the the of for was observed for the for the was with a Km of for was the second the with a of a of μm, the pNP was of low the with the a pNP with the of Km μm, decreased inhibition as of the of the as a substrate we a of pNP oxidation for a the as of of pNP was a of with a of In a of of inhibition of of inhibition of for with the for a the the low the low for a the at a at a of for for observed In the we the with the of the with was with of at the the for the the of the observed the for was the of for of the this the of the was of the a relatively of the a second rapidly with second a the of the as the a of the for the for a observed for the a as the of the with a of this the the the of this was the for the the of inhibition of the for the for a pNP with a for the substrate the of the of the substrate At the pNP for the for the was the the substrate for with pNP at of pNP was the the pNP of for pNP second pNP a the as the at this In the of substrate at the the pNP at the with as as a the pNP the of pNP the of pNP the a of pNP at the with with was pNP of the a of the pNP but with with at the as observed for the the of the at was the the of the the second the the pNP this with of the of CYP2E1. In with the the pNP a with inhibition the of for pNP the the pNP was at the at the for this the of for the pNP of pNP at the at the at the for the of as observed for the with of pNP In pNP the with the pNP a for oxidation was the the of oxidation at the with pNP the as observed for the of this a of with the of a was observed the of the of the pNP for the at the was the as observed the was the for the pNP pNP the a as observed the of at the with of the the pNP observed the the with the of with the with the as observed for pNP at a of In of the observed the at for the was for of a with the at for this at the the we pNP a of the was the this we substrate inhibition of mechanistic of substrate pNP for In the the of a with substrate the of the for pNP a the the of the of the of the the the of for the the of at the of a second at the for the for the of relatively In the a rate of pNP was observed at a rate at the for substrate the observed a a of pNP a the Km μm, In we a of Km of the a observed study with the the the the the of a of the we observed for the of a substrate a second substrate of the with the substrate inhibition the of the of the for substrate the for pNP the of a with with of the was the of oxidation the was with as the with the was observed of this with decreased but Km for pNP at the the of was pNP as of pNP the of the the the pNP as previously of with of pNP was for the of oxidation was the for for we observed the of the the was observed the for we of the the of with pNP for the as inhibition pNP was μm, was the Km for substrate (24 for the the was the of the the the of the of the the the low for CYP2E1. the for this μm, with the the with the as observed for with of the a the for over the the the of of substrate inhibition a second the substrate a the at substrate as the of this second of pNP the was the Km of of the of the pNP we the the for pNP a of of we observed a rate of at pNP of the rate was the observed rate study the as previously min-1) at pNP concentrations the of the the of the the of a of pNP oxidation CYP2E1. pNP concentrations, the the inhibition was the of the of inhibition the of inhibition In inhibition pNP the a for the we the μm, of the of the for pNP at the the of inhibition of of a we as a with this for we a second for pNP In the second pNP the substrate the as as a the the the of the substrate at the with for pNP oxidation was the the was with of the a the the the a the of the at pNP concentrations μm, the of pNP the at the the pNP with the of of pNP was the of pNP the the pNP the of the pNP the at the pNP the of for the pNP with the the for the of with the of a was observed the of the of the pNP In of the at the with the of for pNP oxidation a was a pNP a of In the of the inhibition at decreased the observed the of for the the was a of In the of with CYP2E1. the a with In of this the with the of at the of pNP the a of the the the substrate at the as of for for the of the a a substrate of the of pNP for a the was with of a the of the substrate with with the was the of for the of the for this for the In the of the of the at the was a pNP a of the of the the of pNP oxidation the of for nevertheless, of this with the the the of inhibition for the of the for substrate the was of CYP2E1. the of concentrations of the for the interpretation of the of the of for the of the of the of the of for a a a of of the of of for as the the of of the of as of with the for the for for of as substrate the this a of the p-nitrophenol a of CYP2E1. the a pNP for a of for the a p-nitrophenol p-nitrophenol a the of substrate inhibition for pNP of the the pNP a the observed rate of a was the rate gradually decreased at pNP the was a substrate inhibition the the substrate inhibition of a second with the substrate concentrations this decreased for the of for was inhibition with of a of pNP the of a for the the for was of was the of concentrations of pNP for the of CYP2E1. we this of mechanistic of we as for oxidation of the of inhibition In as the of the a of the CYP2E1. pNP was for of the pNP the the of we for the the the a of a we the of of with a of a second for a the the of the for was was of the In In with was as a pNP for oxidation of pNP a was with a pNP was the the of of with a was the concentrations of a second for of for the the of the for concentrations for this of at for of a was at a with was a low a the for the previously of a with a at a rate of of the pNP at the was concentrations as a of the rate was with the of the observed substrate inhibition pNP oxidation for for substrate nevertheless, of inhibition the for the at the the substrate substrate was the of for In substrate of the substrate substrate but substrate the was the In this the substrate substrate In the of inhibition the the the for inhibition the of as the for this the of for of the of the of a for In this we the the the low for the of the of the the decreased the as a of for the of with for observed we with of at was the a In the we the with the of the with for with the the of a the observed substrate inhibition for pNP oxidation the of was the of with we the as for of the we we the a as the for this for of was the the of the a for the the of the the with the was of the the of CYP2E1. a the the a of the of substrate the for was was of the In In with was as a pNP for oxidation of pNP a was with a pNP was the the of of with a was the concentrations of a second for of for the the of the for concentrations for this of at for of a was at a with was a low a the for the previously of a with a at a rate of of the pNP at the was concentrations as a of the rate was with the of the observed substrate inhibition pNP oxidation for for substrate nevertheless, of inhibition the for the at the the substrate substrate was the of for In substrate of the substrate substrate but substrate the was the In this the substrate substrate In the of inhibition the the the for inhibition the of as the for this the of for of the of the of a for In this we the the the low for the of the of the the decreased the as a of for the of with for observed we with of at was the a In the we the with the of the with for with the the of a the observed substrate inhibition for pNP oxidation the of was the of with we the as for of the we we the a as the for this of was the the of the a for the the of the the with the was of the the of CYP2E1. a the the a of the of substrate previously oxidation of pNP substrate substrate a a the for a substrate substrate we the a as a relatively low (24 the of for nevertheless, at pNP concentrations the gradually decreased as a second at the of was inhibition of of for pNP a of for we the of inhibition of the observed substrate inhibition pNP oxidation for inhibition for substrate nevertheless, of inhibition substrate we a the the of the as a the for the the for for the was a for for In this pNP for the at the of the the concentrations of the for the second we a second of of the over with as for inhibition of pNP inhibition a of for inhibition of pNP of a the for the the of for was observed for the for the was with a Km of for was the second the with a of a of μm, the pNP was of low the with the a pNP with the of Km μm, decreased inhibition as of the of the as a substrate we a of pNP oxidation for a the as of of pNP was a of with a of In a of of inhibition of of inhibition of for with the for a the the low the low for a the at a at a of for for observed In the we the with the of the with was with of at the the for the the of the observed the for was the of for of the this the of the was of the a relatively of the a second rapidly with second a the of the as the a of the for the for a observed for the a as the of the with a of this the the the of this was the for the the of inhibition of the for the for a pNP with a for the substrate the of the of the substrate At the pNP for the for the was the the substrate for with pNP at of pNP was the the pNP of for pNP second pNP a the as the at this In the of substrate at the the pNP at the with as as a the pNP the of pNP the of pNP the a of pNP at the with with was pNP of the a of the pNP but with with at the as observed for the the of the at was the the of the the second the the pNP this with of the of CYP2E1. In with the the pNP a with inhibition the of for pNP the the pNP was at the at the for this the of for the pNP of pNP at the at the at the for the of as observed for the with of pNP In pNP the with the pNP a for oxidation was the the of oxidation at the with pNP the as observed for the of this a of with the of a was observed the of the of the pNP for the at the was the as observed the was the for the pNP pNP the a as observed the of at the with of the the pNP observed the the with the of with the with the as observed for pNP at a of In of the observed the at for the was for of a with the at for this at the the we pNP a of the was the previously oxidation of pNP substrate substrate a a the for a substrate substrate we the a as a relatively low (24 the of for nevertheless, at pNP concentrations the gradually decreased as a second at the of was inhibition of of of for we the of inhibition of the observed substrate inhibition pNP oxidation for inhibition for substrate nevertheless, of inhibition substrate we a the the of the as a the for the the for for the was a for for In this pNP for the at the of the the concentrations of the for the second we a second of of the over with as the for the the of for was observed for the for the was with a Km of for was the second the with a of a of μm, the pNP was of low the with the a pNP with the of Km μm, decreased inhibition as of the of the as a substrate we a of pNP oxidation for a the as of of pNP was a of with a of In a of of inhibition of of inhibition of for with the for a the the low the low for a the at a at a of observed the for was the of for of the this the of the was of the a relatively of the a second rapidly with second a the of the as the a of the for the observed for the a as the of the with a of this the the the of this was the for the the of inhibition of the for the pNP with a for the substrate the of the of the substrate At the pNP for the for the was the the substrate for with pNP at of pNP was the the pNP second pNP a the as the at this In the of substrate at the the pNP at the with as as a the pNP the of pNP the of pNP the a of pNP at the with with was pNP of the a of the pNP but with with at the as observed for the the of the at was the the of the the second the the pNP this with of the of CYP2E1. In with the the pNP a with inhibition the of for pNP the the pNP was at the at the for this the of for the pNP of pNP at the at the at the for the of as observed for the with of pNP In pNP the with the pNP a for oxidation was the the of oxidation at the with pNP the as observed for the of this a of with the of a was observed the of the of the pNP for the at the was the as observed the was the for the pNP pNP the a as observed the of at the with of the the pNP observed the the with the of with the with the as observed for pNP at a of In of the observed the at for the was for of a with the at for this at the the we pNP a of the was the this we substrate inhibition of mechanistic of substrate pNP for In the the of a with substrate the of the for pNP a the the of the of the of the the the of for the the of at the of a second at the for the for the of relatively In the a rate of pNP was observed at a rate at the for substrate the observed a a of pNP a the Km μm, In we a of Km of the a observed study with the the the the the of a of the we observed for the of a substrate a second substrate of the with the substrate inhibition the of the of the for substrate the for pNP the of a with with of the was the of oxidation the was with as the with the was observed of this with decreased but Km for pNP at the the of was pNP as of pNP the of the the the pNP as previously of with of pNP was for the of oxidation was the for for we observed the of the the was observed the for we of the the of with pNP for the as inhibition pNP was μm, was the Km for substrate (24 for the the was the of the the the of the of the the the low for CYP2E1. the for this μm, with the the with the as observed for with of the a the for over the the the of of substrate inhibition a second the substrate a the at substrate as the of this second of pNP the was the Km of of the of the pNP we the the for pNP a of of we observed a rate of at pNP of the rate was the observed rate study the as previously min-1) at pNP concentrations the of the the of the the of a of pNP oxidation CYP2E1. pNP concentrations, the the inhibition was the of the of inhibition the of inhibition In inhibition pNP the a for the we the μm, of the of the for pNP at the the of inhibition of of a we as a with this for we a second for pNP In the second pNP the substrate the as as a the the the of the substrate at the with for pNP oxidation was the the was with of the a the the the a the of the at pNP concentrations μm, the of pNP the at the the pNP with the of of pNP was the of pNP the the pNP the of the pNP the at the pNP the of for the pNP with the the for the of with the of a was observed the of the of the pNP In of the at the with the of for pNP oxidation a was a pNP a of In the of the inhibition at decreased the observed the of for the the was a of In the of with CYP2E1. the a with In of this the with the of at the of pNP the a of the the the substrate at the as of for for the of the a a substrate of the of pNP for a the was with of a the of the substrate with with the was the of for the of the for this for the In the of the of the at the was a pNP a of the of the the of pNP oxidation the of for nevertheless, of this with the the the of inhibition for the of the for substrate the was of CYP2E1. the of concentrations of the for the interpretation of the of the of for the of the of the of the of In this we substrate inhibition of mechanistic of substrate pNP for In the the of a with substrate the of the for pNP a the the of the of the of the the the of for the the of at the of a second at the for the for the of relatively In the a rate of pNP was observed at a rate at the for substrate the observed a a of pNP a the Km μm, In we a of Km of the a observed study with the the the the the of a of the we observed for the of a substrate a second substrate of the with the substrate inhibition the of the of the for substrate CYP2E1. the for pNP the of a with with of the was the of oxidation the was with as the with the was observed of this with decreased but Km for pNP at the the of was pNP as of pNP the of the the the pNP as previously of with of pNP was for the of oxidation was the for for we observed the of the the was observed the for we of the the of with pNP for the as inhibition pNP was μm, was the Km for substrate (24 for the the was the of the the the of the of the the the low for CYP2E1. the for this μm, with the the with the as observed for with of the a the for over the the the of of substrate inhibition a second the substrate a the at substrate as the of this second of pNP the was the Km of of the of the pNP we the the for pNP a of of we observed a rate of at pNP of the rate was the observed rate study the as previously min-1) at pNP concentrations the of the the of CYP2E1. the the of a of pNP oxidation CYP2E1. pNP concentrations, the the inhibition was the of the of inhibition the of inhibition In inhibition pNP the a for the we the μm, of the of the for pNP at the the of inhibition of of a we as a with this for we a second for pNP In the second pNP the substrate the as as a the the the of the substrate at the with for pNP oxidation was the the was with of the a the the the a the of the at pNP concentrations μm, the of pNP the at the the pNP with the of of pNP was the of pNP the the pNP the of the pNP the at the pNP the of for the pNP with the the for the of with the of a was observed the of the of the pNP In of the at the with the of for pNP oxidation a was a pNP a of In the of the inhibition at decreased the observed the of for the the was a of In the of with CYP2E1. the a with In of this the with the of at the of pNP the a of the the the substrate at the as of for for the of the a a substrate of the of pNP for a the was with of a the of the substrate with with the was the of for the of the for this for the In the of the of the at the was a pNP a of the of the the of pNP oxidation the of for nevertheless, of this with the the the of inhibition for the of the for substrate the was of CYP2E1. the of concentrations of the for the interpretation of the of the of for the of the of the of the of for the of for the this with with substrate of