Significant increase in phenacetin oxidation on L382V substitution

ABSTRACT: Human CYP1A2 is an important drug-metabolizing enzyme, similar in sequence to CYP1A1 but with distinct substrate specificity. We have previously shown that residue 382 affected CYP1A1 and CYP1A2 specificities with alkoxyresorufins. To determine whether this residue is also important for the metabolism of other substrates, we have investigated phenacetin oxidation by single (T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/ T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/ N312L) mutants of CYP1A2. The enzymes were expressed in Escherichia coli and purified. All the CYP1A2 mutants that contained the L382V substitution displayed much higher activities than the wildtype enzyme, with k cat values 3-fold higher, in contrast to other mutants, for which k cat decreased. Likewise, a significant increase in specificity, expressed as the k cat /K m ratio, was observed for the mutants containing the L382V substitution. The efficiency of coupling of reducing equivalents to acetaminophen formation was decreased for all the single mutants except L382V, for which the coupling increased. This effect was also observed with multiple CYP1A2 mutants containing the L382V substitution. Low activities of the four other single mutants were likely caused by dramatically increased uncoupling to water. In contrast, the increase in activity of the L382V-containing mutants resulted from decreased water formation. This finding is consistent with molecular dynamics results, which showed decreased phenacetin mobility leading to increased product formation. The results of these studies confirm the importance of residue 382 in CYP1A2-catalyzed oxidations and show that a single residue substitution can dramatically affect enzymatic activity. Cytochromes P450 (P450s) are heme-containing monooxygenase enzymes, which are involved in the metabolism of numerous exogenous and endogenous compounds. P450s are ubiquitous in living organisms, with at least 50 families and 82 subfamilies found in different species. Human CYP1A subfamily has two major isoforms: CYP1A1 and CYP1A2. CYP1A2, one of the major P450s in the human liver, was first characterized as a phenacetin O-deethylase In humans, CYP1A2 shares 72% amino acid sequence identity with CYP1A1, but the substrate specificities and inhibitor susceptibilities of these enzymes are different. For example, substrates such as phenacetin and 7-methoxyresorufin are primarily metabolized by CYP1A2 with high catalytic efficiency, whereas CYP1A1 displays weak capability to oxidize those substrates. On the other hand, 7-ethoxyresorufin is preferentially oxidized by CYP1A1 Our previous studies on structure-function relationships of CYP1A1 indicated that Val382 played an important role in binding of alkoxyresorufin substrates In the current study, we chose phenacetin as a substrate. This compound has been used as the most common marker for CYP1A

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism s

ABSTRACT Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structurefunction relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates-namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarinswith a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (k cat /K M ) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl) coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl) coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B-4-CPI-bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple -Cl and -NH-p interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships