Spectral and kinetic studies on the formation of myeloperoxidase compounds I and II: roles of hydrogen peroxide and superoxide.
The conversion of myeloperoxidase to compounds I and II in the presence of H2O2 has been reinvestigated in order to explain the abnormal stoichiometry of compound I formation and the fast spontaneous decay of compound I to compound II. Rapid-scan studies show that at least a 20-fold excess of H2O2 is required to obtain a good spectrum of relatively pure compound I; a further increase in H2O2 concentration causes compound I to be reduced to compound II, which is a very stable intermediate. Compound I formation is reversible, with an apparent second-order forward rate constant of (1.8 +/- 0.1) x 10(7) M-1 s-1 and a reverse rate constant of 58 +/- 4 s-1, giving a constant of 3.2 microM for the dissociation of compound I to native enzyme and H2O. This reversibility is one factor that can explain the large excess of H2O2 required to form compound I. The apparent second-order rate constant for compound II formation from compound I and H2O2 is (8.2 +/- 0.2) x 10(4) M-1 s-1. We confirm pH dependence studies, which suggest that the formation of compounds I and II is controlled by a residue in the enzyme with a pKa of about 4.0. Excess H2O2 is also converted to O2 via catalase activity of the enzyme. However, we do not consider this a dominant pathway because it fails to account for the fast spontaneous reduction of compound I to compound II. The time courses for both the decay of compound I and the formation of compound II are biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)
Marquez
L A
LA
Department of Chemistry, University of Alberta, Edmonton, Canada.
Huang
J T
JT
Dunford
H B
HB
D000818:Animals; D002417:Cattle; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism; D011190:Potassium Cyanide / Q000378:metabolism; D013053:Spectrophotometry; D013154:Spleen / Q000201:enzymology; D013481:Superoxides / Q000494:pharmacology*
D013481:Superoxides; D006861:Hydrogen Peroxide; D009195:Peroxidase; D011190:Potassium Cyanide