A steady-state study on the formation of Compounds II and III of myeloperoxidase.
The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.
Hoogland
H
H
Laboratory of Biochemistry, University of Amsterdam, The Netherlands.
Dekker
H L
HL
van Riel
C
C
van Kuilenburg
A
A
Muijsers
A O
AO
Wever
R
R
D000465:Algorithms; D005295:Ferrocyanides / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007962:Leukocytes / Q000201:enzymology; D009195:Peroxidase / Q000378:metabolism*; D013053:Spectrophotometry; D013997:Time Factors
D005295:Ferrocyanides; D006861:Hydrogen Peroxide; D009195:Peroxidase; C020354:hexacyanoferrate II