PT - JOURNAL ARTICLE AU - Qingbiao Huang AU - Rahul S. Deshmukh AU - Spencer S. Ericksen AU - Youbin Tu AU - Grazyna D. Szklarz TI - Preferred Binding Orientations of Phenacetin in CYP1A1 and CYP1A2 Are Associated with Isoform-Selective Metabolism AID - 10.1124/dmd.112.047308 DP - 2012 Dec 01 TA - Drug Metabolism and Disposition PG - 2324--2331 VI - 40 IP - 12 4099 - http://dmd.aspetjournals.org/content/40/12/2324.short 4100 - http://dmd.aspetjournals.org/content/40/12/2324.full SO - Drug Metab Dispos2012 Dec 01; 40 AB - Human cytochromes P450 1A1 and 1A2 play important roles in drug metabolism and chemical carcinogenesis. Although these two enzymes share high sequence identity, they display different substrate specificities and inhibitor susceptibilities. In the present studies, we investigated the structural basis for these differences with phenacetin as a probe using a number of complementary approaches, such as enzyme kinetics, stoichiometric assays, NMR, and molecular modeling. Kinetic and stoichiometric analyses revealed that substrate specificity (kcat/Km) of CYP1A2 was approximately 18-fold greater than that of CYP1A1, as expected. Moreover, despite higher H2O2 production, the coupling efficiency of reducing equivalents to acetaminophen formation in CYP1A2 was tighter than that in CYP1A1. CYP1A1, in contrast to CYP1A2, displayed much higher uncoupling, producing more water. The subsequent NMR longitudinal (T1) relaxation studies with the substrate phenacetin and its product acetaminophen showed that both compounds displayed similar binding orientations within the active site of CYP1A1 and CYP1A2. However, the distance between the OCH2 protons of the ethoxy group (site of phenacetin O-deethylation) and the heme iron was 1.5 Å shorter in CYP1A2 than in CYP1A1. The NMR findings are thus consistent with our kinetic and stoichiometric results, providing a likely molecular basis for more efficient metabolism of phenacetin by CYP1A2.