Kinetic isotope effects implicate the iron-oxene as the sole oxidant in P450-catalyzed N-dealkylation.

Multiple oxidants have been implicated as playing a role in cytochrome P450-mediated oxidations. Herein, we report results on N-dealkylation, one of the most facile reactions mediated by P450 enzymes. We have employed the N-oxides of a series of para-substituted 13C2H2-labeled N,N-dimethylanilines to function as both substrates and surrogate oxygen atom donors for P450cam and P4502E1. Kinetic isotope effect profiles obtained using the N-oxide system were found to closely match the profiles produced using the complete NAD(P)H/NAD(P)-P450 reductase/O2 system. The results are consistent with oxidation occurring solely through an iron-oxene species.

Quantitative binding models for CYP2C9 based on benzbromarone analogues.

The cytochrome P450 (CYP) isoforms involved in xenobiotic metabolism are enzymes whose substrate selectivity remains difficult to predict due to wide specificity and dynamic protein-substrate interactions. To uncover the determinants of specificity for cytochrome CYP2C9, a novel library of benzbromarone (bzbr) inhibitors was used to reevaluate its pharmacophore. CoMSIA was used with the bzbr ligands to generate both quantitative binding models and three-dimensional contour plots that pinpoint predicted interactions that are important for binding to 2C9. Since this class of compounds is more potent than any other toward 2C9, the small molecule properties deemed most ideal by the software were used to address protein-ligand interactions using new mutagenesis and structural data. Nine new bzbr analogues provide evidence that specific electrostatic and hydrophobic interactions contribute the most to 2C9's specificity. Three of the new analogues are better isosteres of bzbr that contain bulky groups adjacent to the phenol and have increased pK(a) values. These ligands test the hypothesis that anionic substrates bind with higher affinity to 2C9. Since they have higher affinity than the previous nonacidic analogues, the importance of bulky groups on the phenol ring appears to have been underestimated. CoMSIA models predict that these bulky groups are favorable for their hydrophobicity, while a negative charge is favored at the ketone oxygen rather than the phenol oxygen. The overlap of this ketone with electronegative groups of other 2C9 substrates suggests they act as key positive charge acceptors.

An analysis of the regioselectivity of aromatic hydroxylation and N-oxygenation by cytochrome P450 enzymes.

Quinoline was used to probe the steric and electronic contributions to rates of aromatic oxidation of nitrogen-containing, multiring substrates by cytochrome P450 (P450) enzymes. The regioselectivity of the P450 oxidation of quinoline was determined experimentally by identifying and measuring the ratios of metabolites. The laboratory results were compared with those obtained computationally by modeling the electronic effects for aromatic hydroxylation of the substrate. Calculated values predict 8-hydroxyquinoline to have the lowest relative activation energy, whereas 3-hydroxyquinoline was calculated to have the highest relative activation energy. In contrast, 3-hydroxyquinoline was produced to a much greater extent relative to 8-hydroxyquinoline. The sharp contrast observed between the computationally obtained energies and the ratios of products identified experimentally indicates that steric factors play a role in determining the regioselectivity of P450 enzymes with quinoline. To further probe steric contributions to product formation, isoquinoline was used as a substrate and the results were compared with those obtained with quinoline. Isoquinoline N-oxide was determined to be the major metabolite of isoquinoline with all of the P450 enzymes used. These results provide further evidence for the steric influence on the regioselectivity of P450 enzymes with quinoline.

A new class of CYP2C9 inhibitors: probing 2C9 specificity with high-affinity benzbromarone derivatives.

Noncovalent forces, other than hydrophobic interactions, are important determinants of substrate bias exhibited by some cytochromes P450. The CYP2C9 pharmacophore is proposed to include either an anionic group or hydrogen bond donor in addition to its hydrophobic groups. By constructing analogs of benzbromarone, evidence supporting the existence of a 2C9 anion-binding site was revealed. A nonsubstituted phenol analog was determined to have a pKa of 8.4 and a Ki of 414 nM whereas those with dihalogenated benzoyl phenols had pKa values between 4.2 to 5.2 and Ki values as low as 1 nM. The nonhalogenated, nonionizable analog is the poorest binder at 796 nM. The Ki range covers around three orders of magnitude with even the weakest binder being a more potent inhibitor than 2C9 substrate phenytoin. Thus, benzbromarone derivatives represent a class of molecules with the potential to reveal more structural details of the 2C9 active site.

Evidence for two different active oxygen species in cytochrome P450 BM3 mediated sulfoxidation and N-dealkylation reactions.

Herein, we report the results from two experiments that are consistent with sulfoxidation and N-dealkylation involving two different enzyme substrate complexes and thus two different active oxygen species that do not interchange. The first experiment involves the use of a mutant that may increase the amount of the hydroperoxy-iron species (FeIIIO2H).1 This mutant increases the amount of sulfoxidation relative to the amount of N-dealkylation by 4-fold. In a second experiment, deuterium substitution on the N-methyl groups of substrate does not result in an increase in sulfoxidation. This later result is consistent with N-dealkylation and sulfoxidation being mediated by two different active oxygen species. While the data indicate two active oxygen species, they do not distinguish between the different possibilities for the active oxygen species.

Use of kinetic isotope effects to delineate the role of phenylalanine 87 in P450(BM-3).

The substrate oxidation rates of P450(BM-3) are unparalleled in the cytochrome P450 (CYP) superfamily of enzymes. Furthermore, the bacterial enzyme, originating from Bacillus megaterium, has been used repeatedly as a model to study the metabolism of mammalian P450s. A specific example is presented where studying P450(BM-3) substrate dynamics can define important enzyme-substrate characteristics, which may be useful in modeling omega-hydroxylation seen in mammalian P450s. In addition, if the reactive species responsible for metabolism can be controlled to produce specific products this enzyme could be a useful biocatalyst. Based on crystal structures and the fact that the P450(BM-3) F87A mutant produces a large isotope in contrast to the native enzyme, we propose that phenylalanine 87 is responsible for hindering substrate access to the active oxygen species for nonnative substrates. Using kinetic isotopes and two aromatic substrates, p-xylene and 4,4'-dimethylbiphenyl, the role phenylalanine 87 plays in active-site dynamics is characterized. The intrinsic KIE is 7.3 +/- 2 for wtP450(BM-3) metabolism of p-xylene. In addition, stoichiometry differences were measured with the native and mutant enzyme and 4,4'-dimethylbiphenyl. The results show a more highly coupled substrate/NADPH ratio in the mutant than in the wtP450(BM-3).

Active site mutations of cytochrome p450cam alter the binding, coupling, and oxidation of the foreign substrates (R)- and (s)-2-ethylhexanol.

Three factors are of primary importance with respect to designing efficient P450 biocatalysts. (1) The substrate must be oxidized at a significant rate. (2) The regioselectivity must heavily favor the desired product. (3) The enzyme must use the majority of the reducing equivalents from NADH or NADPH to produce product. The reaction we chose to study was oxidation of 2-ethylhexanol to 2-ethylhexanoic acid by P450cam. We examined four active site mutations: F87W, Y96W, T185F, and L244A. The mutations were chosen to improve 2-ethyhexanoic acid production by decreasing active site volume, increasing active site hydrophobicity, and improving stereoselectivity. The F87W and Y96W mutations improved regioselectivity, giving almost exclusively the desired product. The T185F mutation improved coupling of NADH to product formation. The L244A mutation altered the stereoselectivity of 2-ethylhexanoic acid production. These results indicate that active site mutations of P450cam can alter catalysis of 2-ethylhexanol.