A transient kinetic study on the reactivity of recombinant unprocessed monomeric myeloperoxidase.
Spectral and kinetic features of the redox intermediates of human recombinant unprocessed monomeric myeloperoxidase (recMPO), purified from an engineered Chinese hamster ovary cell line, were studied by the multi-mixing stopped-flow technique. Both the ferric protein and compounds I and II showed essentially the same kinetic behavior as the mature dimeric protein (MPO) isolated from polymorphonuclear leukocytes. Firstly, hydrogen peroxide mediated both oxidation of ferric recMPO to compound I (1.9 x 10(7) M(-1) s(-1), pH 7 and 15 degrees C) and reduction of compound I to compound II (3.0 x 10(4) M(-1) s(-1), pH 7 and 15 degrees C). With chloride, bromide, iodide and thiocyanate compound I was reduced back to the ferric enzyme (3.6 x 10(4) M(-1) s(-1), 1.4 x 10(6) M(-1) s(-1), 1.4 x 10(7) M(-1) s(-1) and 1.4 x 10(7) M(-1) s(-1), respectively), whereas the endogenous one-electron donor ascorbate mediated transformation of compound I to compound II (2.3 x 10(5) M(-1) s(-1)) and of compound II back to the resting enzyme (5.0 x 10(3) M(-1) s(-1)). Comparing the data of this study with those known from the mature enzyme strongly suggests that the processing of the precursor enzyme (recMPO) into the mature form occurs without structural changes at the active site and that the subunits in the mature dimeric enzyme work independently.
Furtmüller
P G
PG
Institute of Chemistry, University of Agricultural Sciences, Vienna, Austria.
Jantschko
W
W
Regelsberger
G
G
Jakopitsch
C
C
Moguilevsky
N
N
Obinger
C
C
D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain; D006224:Cricetinae; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006997:Hypochlorous Acid / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D009195:Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D009195:Peroxidase