title: Mechanistic evaluation of substrate inhibition kinetics observed from aldehyde oxidase-catalyzed reactions.
abstract: While most enzyme-catalyzed reactions are adequately described by Michaelis-Menten kinetics, Aldehyde Oxidase (AOX) metabolism might exhibit atypical kinetics due to possible substrate inhibition. Ignoring this phenomenon may lead to erroneous estimates of kinetic parameters and over simplification of the enzyme mechanism. In this study, in vitro metabolism data for 3 AOX substrates exhibiting varying degrees of substrate inhibition were analyzed with the following kinetic models: A) Michaelis-Menten (naïve) model; B) Substrate inhibition (empirical) model; and C) Twobinding site (mechanistic) model. The application of this mechanistic model is a novel interpretation for kinetic analysis of AOX metabolism whereby substrate can presumably bind to two enzymes' active site(s). Unlike the other models, this mechanistic model quantitatively captures the degree of substrate inhibition observed. Analysis by this model showed: A) All tested substrates have simultaneous access to the metabolic and inhibitory site of the enzyme with Ks (binding affinity for inhibitory site) greater (1.3- to 28-fold) than Km (binding affinity for metabolic site); B) Dissociation constants for binding of a second substrate in either the productive and nonproductive enzyme conformations decreased with factor α ranging from 2.58 to 15.6 between compounds; and C) In addition, a drastic decrease (from 64%-98%) in the metabolism rates between compounds was exhibited by factor β (ranging from 0.02-0.36). Overall, the mechanistic two-binding site model best fitted the experimental data. Moreover, the observed differences between kinetic parameters generated by these models highlight the importance of appropriate model selection to adequately fit the substrate inhibition kinetics of AOX metabolism.
authors.forename: Stephen W J
authors.initials: SW
authors.lastname: Wang
authors.forename: Kojo
authors.initials: K
authors.lastname: Abdul-Hadi
authors.forename: Lawrence
authors.initials: L
authors.lastname: Cohen
authors.affiliation: 35 Lansdowne Street, Drug Metabolism and Pharmacokinetics, Millennium Pharmaceuticals, Inc. Cambridge, MA 02139 USA. stephen.wang@mpi.com.
authors.forename: Cindy Q
authors.initials: CQ
authors.lastname: Xia
mesh_terms: D000212:Acyclovir / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D042931:Aldehyde Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D000465:Algorithms; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006146:Guanidines / Q000493:pharmacokinetics; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D015233:Models, Statistical; D010793:Phthalazines / Q000378:metabolism; D011720:Pyrazoles / Q000493:pharmacokinetics; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C108016:6-deoxypenciclovir; D004791:Enzyme Inhibitors; D006146:Guanidines; D010793:Phthalazines; D011720:Pyrazoles; C424422:zoniporide; D042931:Aldehyde Oxidase; D000212:Acyclovir