INTERACTION OF MYELOPEROXIDASE WITH PEROXYNITRITE - A COMPARISON WITHLACTOPEROXIDASE, HORSERADISH-PEROXIDASE AND CATALASE

Polymorphonuclear neutrophils generate both nitric oxide and superoxid e and these molecules can combine to form peroxynitrite. Neutrophils a lso contain myeloperoxidase which reacts with peroxynitrous acid (HOON O). On mixing myeloperoxidase with HOONO compound II was formed. Compo und I could not be detected as an intermediate. The apparent second-or der rate constant of formation of compound II was strongly pH-dependen t (2.5 X 10(5) M-1 . s-1 at pH 8.9 and 6.2 X 10(6) M-1 . s-1 at pH 7.2 ). The pK(a) of this effect is 6.9 and it was concluded that the enzym e reacts with the protonated form of the peroxide, that is peroxynitro us acid, with a pH-independent second-order rate constant of 2.0 X 10( 7) M-1 . s-1 at 12-degrees-C. The interaction of HOONO with lactoperox idase was studied for comparison. As was observed for myeloperoxidase, compound I could not be detected as an intermediate. The apparent sec ond-order rate constant of compound II formation is pH-dependent and i s 3.3 X 10(5) M-1 . s-1 at pH 7.4 and 8.4 X 10(4) M-1 . s- 1 at pH 9.0 . In contrast, horseradish peroxidase reacts with HOONO to form compou nd 1, which is subsequently followed by the formation of compound II. The second-order rate constant for the formation of compound I is 3.2 X 10(6) M-1 . s-1 and is pH-dependent, the pK(a) for this effect is 6. 8. Catalase (up to 3 muM) does not affect the rate of decomposition of peroxynitrite and no compound I formation is observed. Since nitrite may be present in the peroxynitrite preparation and to discriminate be tween the reaction of the enzyme with nitrite or peroxynitrite, the ef fect of nitrite on myeloperoxidase was studied. The dissociation const ant for the myeloperoxidase-nitrite complex is pH-dependent and has va lues of 580 muM at pH 6.0 and 55 mM at pH 8.5.