Inactivation of human myeloperoxidase by oxidants and suicide substrates

Myeloperoxidase (MPO) is abundantly present in neutrophils and participates in the innate immune defence through the formation of reactive oxidants and diffusible radical species. It is a heme peroxidase that uses hydrogen peroxide (H2 O2 ) and halides (chloride and bromide) to produce hypohalous acids (HOCl and HOBr), which are potent antimicrobial agents. However, MPO derived oxidants have also been implicated in promoting tissue damage in numerous inflammatory diseases, which has attracted considerable interest in the development of therapeutically useful MPO inhibitors. MPO is known to be inactivated during the production of reactive oxidants. Both, H 2 O2 and HOCl alone were found to inactivate the enzyme as well. In the course of inactivation MPO is likely to undergo oxidation and halogenation reactions, which lead to modifications of amino acid residues and possibly of the heme prosthetic groups. In unpublished work it was found that oxidatively modified MPO kills bacteria (see preliminary results). The required enzyme modifications for killing have yet to be revealed. Such modifications of MPO may be useful biomarkers of oxidant production in vivo and assist in evaluating the contribution the enzyme makes to various pathologies. Promising suicide inhibitors have been described to irreversibly inhibit MPO, however, the modifications the enzyme undergoes are unknown and have not been addressed on a structural basis. The specific goal of this proposal is to investigate the mechanisms and sites of enzyme modification, which lead to MPO inactivation during oxidant generation by stimulated neutrophils as well as when the enzyme is exposed to the relevant oxidants. Furthermore the sites of modification when reacting with suicide substrates will be investigated. By revealing the mechanisms and sites of modification and their relatedness to enzyme inactivation, valuable information can be gained for the design of highly specific MPO-inhibitors as well as for the development of specific biomarkers for oxidized or inactivated MPO in biological fluids, which will allow for a more direct assessment of the role of MPO in disease. Mature dimeric MPO purified from human neutrophils will be incubated with the relevant oxidants at various defined conditions and time intervals. Steady-state and pre-steady state kinetic analysis will be performed in combination with various liquid chromatography/mass spectrometric techniques to identify amino acid residue and heme modifications. Their effects on the halogenation and peroxidase activity will be investigated.