TY - JOUR T1 - Site Directed Mutagenesis at the MoCo Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.120.000187 SP - DMD-AR-2020-000187 AU - Armina Abbasi AU - Carolyn A. Joswig-Jones AU - Jeffrey P. Jones Y1 - 2020/01/01 UR - http://dmd.aspetjournals.org/content/early/2020/10/08/dmd.120.000187.abstract N2 - The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since humans (hAO) and cynomolgus monkeys (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO respectively, to compare and contrast the kinetics of these two species through numerical modeling. While dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor (MoCo) to the corresponding alanine and leucine in mAO respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, six more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. Significance Statement Even with having as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans. ER -