TY - JOUR T1 - Significant Increase in Phenacetin Oxidation on L382V Substitution in Human Cytochrome P450 1A2 JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1039 LP - 1045 DO - 10.1124/dmd.109.030767 VL - 38 IS - 7 AU - Qingbiao Huang AU - Grazyna D. Szklarz Y1 - 2010/07/01 UR - http://dmd.aspetjournals.org/content/38/7/1039.abstract N2 - 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 wild-type enzyme, with kcat values 3-fold higher, in contrast to other mutants, for which kcat decreased. Likewise, a significant increase in specificity, expressed as the kcat/Km 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. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics ER -