Ben G.J.M. Bolscher

Some properties of human eosinophil peroxidase, a comparison with other peroxidases.

Eosinophil peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) was isolated from outdated human white blood cells. The purified enzyme has a molecular weight of 71000 +/- 1000. The enzyme is composed of two subunits, of Mr 58000 and 14000, in a 1:1 stoichiometry. Amino-acid analyses showed that eosinophil peroxidase has a high content of the amino acids arginine, leucine and aspartic acid. The millimolar absorbance coefficient of the Soret band at 412 nm of eosinophil peroxidase was determined. Three independent methods yield a value for epsilon 412nm of 110 +/- 4 mm-1 X cm-1. Purified eosinophil peroxidase showed a homogeneous high-spin EPR signal with rhombic symmetry (gx = 6.50; gy = 5.40; gz = 1.982) for the haem group. EPR spectroscopy of low-spin cyanide and azide derivatives of eosinophil peroxidase, lactoperoxidase, myeloperoxidase and catalase revealed that the haem-ligand structure of eosinophil peroxidase is closely related to lactoperoxidase, whereas that of myeloperoxidase shows great resemblance to catalase.

A kinetic study of the reaction between human myeloperoxidase, hydroperoxides and cyanide inhibition by chloride and thiocyanate

The reaction between myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), hydrogen peroxide and ethyl hydroperoxide was investigated using the stopped-flow technique. Like other peroxidases, myeloperoxidase forms two sequential peroxide compounds. The pH-dependence of the apparent second-order rate constant of compound I formation shows that there is an acid/base group on the enzyme with a pKa of 4.30 +/- 0.15, which - when protonated - prevents the reaction of the enzyme with peroxides. The rate constants for the formation of compound I by hydrogen peroxide and ethyl hydroperoxide are (2.3 +/- 0.1) X 10(7) M-1 X s-1 and (2.8 +/- 0.3) X 10(5) M-1 X s-1, respectively. The binding of cyanide to myeloperoxidase (k1 = (1.30 +/- 0.05) X 10(6) M-1 X s-1) is also regulated by an acid/base group with a pKa of 4.00 +/- 0.05 as is the case with hydrogen peroxide; also, only the protonated uncharged form of cyanide reacts with the enzyme. From their effects on the binding of cyanide to the enzyme it is concluded that chloride and thiocyanate bind to myeloperoxidase only when the acid/base group is protonated. The pH-dependence of the dissociation constant of the myeloperoxidase-chloride complex obtained from the spectral changes induced by chloride is the same as observed in the inhibition by chloride of the binding of cyanide. It is concluded that hydrogen peroxide, cyanide, chloride and thiocyanate bind at the same site on the enzyme.

A Point Mutation in gp91-phox of Cytochrome b558 of the Human NADPH Oxidase Leading to Defective Translocation of the Cytosolic Proteins p47-phox and p67-phox

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patients neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.

Vitamin C stimulates the chlorinating activity of human myeloperoxidase

Ascorbic acid (vitamin C) was found to stimulate the chlorinating activity of human myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) 3-fold in vitro and to shift the pH optimum of the reaction to higher pH values. These effects are due to the conversion by ascorbic acid of inactive compound II formed during turnover into native enzyme.

The nitrosyl compounds of ferrous animal haloperoxidases

Human myeloperoxidase, human eosinophil peroxidase and bovine lactoperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) reduced with ascorbic acid form nitrosyl compounds which show rhombic EPR signals centered at g = 2. Using 14NO (IN = 1), the central resonance signal exhibited a hyperfine structure of nine lines originating from a triplet with a small hyperfine splitting (AII(zeta) = 0.69 mT for myeloperoxidase and 0.73 mT for eosinophil peroxidase and lactoperoxidase) superimposed upon a triplet with a larger hyperfine splitting (AI(zeta) = 2.34, 2.32 and 2.09 mT for myeloperoxidase, eosinophil peroxidase and lactoperoxidase, respectively). Using 15NO (IN = 1/2), the nitrosyl compound of ferrous myeloperoxidase and ferrous lactoperoxidase showed a doublet of triplets superimposed upon the central resonance signal. These results demonstrate that a nitrogen nucleus is present at the fifth ligand position of the haem iron in these peroxidases.

NADPH:O2 oxidoreductase of human eosinophils in the cell‐free system

The NADPH oxidase of human eosinophils, measured in the cell‐free system, shows the same characteristics as the enzyme from human neutrophils. All proteins required for activity of the enzyme are expressed in eosinophils at a higher level than in neutrophils. Eosinophils isolated from patients with chronic granulomatous disease show the same molecular defects as the neutrophils fom these patients.

Generation of reactive oxygen species by phagocytes

Phagocytic leukocytes (neutrophilic granulocytes, eosinophilic granulocytes, monocytes and macrophages) are effector cells in our defense against microbial pathogens. Phagocytes kill a variety of microorganisms by ingesting them and attacking them intracellularly with hydrolytic enzymes, bactericidal proteins, reactive oxygen species, and perhaps nitric oxide. This chapter will be restricted to the generation of reactive oxygen species; other microbicidal mechanisms will be dealt with in other chapters.

The Respiratory Burst and the NADPH Oxidase of Phagocytic Leukocytes

When phagocytic leukocytes are activated to kill microorganisms, these cells respond with considerable changes in their cellular metabolism and structure.1 Most marked is a 20- to 30-fold increase in the oxygen consumption (i.e., the respiratory burst) that is not sensitive to inhibitors of the mitochondrial respiration.2 The extra oxygen consumption reflects the action of a phagocyte-specific oxidase, responsible for the generation of reduced oxygen species.1 This enzyme is localized in the plasma membrane and—after phagocytosis—in the phagosomal membranes of the cell.3,4