title: Inhibition of the chlorinating activity of myeloperoxidase by tempol: revisiting the kinetics and mechanisms.
abstract: The nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of inflammation by mechanisms that are not completely understood. MPO (myeloperoxidase), which plays a fundamental role in oxidant production by neutrophils, is an important target for anti-inflammatory action. By amplifying the oxidative potential of H2O2, MPO produces hypochlorous acid and radicals through the oxidizing intermediates MPO-I [MPO-porphyrin•+-Fe(IV)=O] and MPO-II [MPO-porphyrin-Fe(IV)=O]. Previously, we reported that tempol reacts with MPO-I and MPO-II with second-order rate constants similar to those of tyrosine. However, we noticed that tempol inhibits the chlorinating activity of MPO, in contrast with tyrosine. Thus we studied the inhibition of MPO-mediated taurine chlorination by tempol at pH 7.4 and re-determined the kinetic constants of the reactions of tempol with MPO-I (k=3.5×105 M-1·s-1) and MPO-II, the kinetics of which indicated a binding interaction (K=2.0×10-5 M; k=3.6×10-2 s-1). Also, we showed that tempol reacts extremely slowly with hypochlorous acid (k=0.29 and 0.054 M-1·s-1 at pH 5.4 and 7.4 respectively). The results demonstrated that tempol acts mostly as a reversible inhibitor of MPO by trapping it as MPO-II and the MPO-II-tempol complex, which are not within the chlorinating cycle. After turnover, a minor fraction of MPO is irreversibly inactivated, probably due to its reaction with the oxammonium cation resulting from tempol oxidation. Kinetic modelling indicated that taurine reacts with enzyme-bound hypochlorous acid. Our investigation complements a comprehensive study reported while the present study was underway
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil.
authors.forename: Raphael F
authors.initials: RF
authors.lastname: Queiroz
authors.forename: Sandra M
authors.initials: SM
authors.lastname: Vaz
authors.forename: Ohara
authors.initials: O
authors.lastname: Augusto
mesh_terms: D003497:Cyclic N-Oxides / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics; D054879:Halogenation / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D009195:Peroxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000493:pharmacokinetics; D013113:Spin Labels
chemical_list: D003497:Cyclic N-Oxides; D004791:Enzyme Inhibitors; D013113:Spin Labels; D009195:Peroxidase; C001803:tempol