The chlorinating activity of human myeloperoxidase: high initial activity at neutral pH value and activation by electron donors.

The steady-state activity of myeloperoxidase in the chlorination of monochlorodimedone at neutral pH was investigated. Using a stopped-flow spectrophotometer we were able to show that the enzymic activity at pH 7.2 rapidly declined in time. During the first 50-100 ms after addition of H2O2 to the enzyme, a turnover number of about 320 s-1 per haem was observed. However, this activity decreased rapidly to a value of about 25s-1 after 1 s. This shows that in classical steady-state activity measurements, the real activity of the enzyme at neutral pH is grossly underestimated. By following the transient spectra of myeloperoxidase during turnover it was shown that the decrease in activity was probably caused by the formation of an enzymically inactive form of the enzyme, Compound II. As demonstrated before (Bolscher, B.G.J.M., Zoutberg, G.R., Cuperus, R.A. and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191) reductants such as ascorbic acid and ferrocyanide convert Compound II, which accumulates during turnover, into active myeloperoxidase. Activity measurements in the presence of ascorbic acid showed, indeed, that the moderate enzymic activity was higher than in the absence of ascorbic acid. With 5-aminosalicylic acid present, however, the myeloperoxidase activity remained at a much higher level, namely about 150 s-1 per haem during the time interval from 100 ms to 5 s after mixing. From combined stopped-flow/rapid-scan experiments during turnover it became clear that in the presence of 5-aminosalicylic acid the initially formed Compound II was rapidly converted back to native enzyme. Presteady-state experiments showed that 5-aminosalicylic acid reacted with Compound II with a K2 of 3.2 x 10(5) M-1.s-1, whereas for ascorbic acid a K2 of 1.5 x 10(4) M-1.s-1 was measured at pH 7.2. In the presence of 5-aminosalicylic acid during the time interval in which the myeloperoxidase activity remained constant, a Km for H2O2 at pH 7.2 was determined of about 30 microM at 200 mM chloride. In the absence of reductants the same value was found during the first 100 ms after addition of H2O2 to the enzyme. The physiological consequences of these findings are discussed.

Interaction of myeloperoxidase with diclofenac. Inhibition of the chlorinating activity of myeloperoxidase by diclofenac and oxidation of diclofenac to dihydroxyazobenzene by myeloperoxidase.

The chlorinating activity of myeloperoxidase, isolated from human polymorphonuclear neutrophils, was inhibited by the non-steroidal anti-inflammatory drug diclofenac (Voltaren). The concentration of diclofenac needed for 50% inhibition was 20 microM, a value comparable with IC50 values found for other drugs. Diclofenac did not react with HOCl nor with H2O2 but was oxidized in the presence of myeloperoxidase and H2O2 to an orange-coloured unstable product. The rate of oxidation was proportional to the enzyme concentration and to the concentration of diclofenac. but independent of the H2O2 concentration. Presumably both Compound I and Compound II, two intermediates formed during the reaction cycle of myeloperoxidase with H2O2 are able to oxidize diclofenac. In these redox reactions, the active short-living Compound I is reduced to Compound II, thereby inhibiting the chlorinating activity of the enzyme. Analysis by Fast Atom Bombardment mass spectrometry showed that in the presence of H2O2 myeloperoxidase oxidizes diclofenac to dihydroxyazobenzene.

4-Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological substrates.

Valerophenone synthase (VPS) is a polyketide synthase that catalyzes the formation of the phloroglucinol derivatives in the synthesis of the bitter acids in hop (Humulus lupulus). The reaction uses isovaleryl-CoA or isobutyryl-CoA, but otherwise it is identical to that of the chalcone synthase in flavonoid biosynthesis. Our study showed that chalcone synthase can perform the function of VPS, but not perfectly, because the majority of the reactions terminated after two condensation reactions (products: 4-hydroxy-2-pyrone derivatives). The same experiments with stilbene synthase yielded exclusively the 4-hydroxy-2-pyrone derivatives, not the products expected from three condensation reactions. The results are discussed in the context of the functional diversity and evolution in the family of CHS-related polyketide synthases.

Human hemi-myeloperoxidase. Initial chlorinating activity at neutral pH, compound II and III formation, and stability towards hypochlorous acid and high temperature.

Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro-organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide-linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two promoters, 'hemi-myeloperoxidase'. We studied the initial activities of human dimeric MPO and hemi-MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds II and III, both inactive forms concerning HOCl generation, and the rate constants of their formation were the same for dimeric MPO and hemi-MPO, but hemi-MPO required a slightly larger excess of H2O2 for complete conversion. Hemi-MPO was less stable at a high temperature (80 degrees C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi-MPO against its oxidative product hypochlorous acid was somewhat lower (IC50 = 32 microM HOCl) compared to dimeric MPO (IC50 = 50 microM HOCl). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.

Phlorisovalerophenone synthase, a novel polyketide synthase from hop (Humulus lupulus L.) cones.

Phlorisovalerophenone synthase (VPS), a novel aromatic polyketide synthase, was purified to homogeneity from 4.2 mg protein extract obtained from hop (Humulus lupulus L.) cone glandular hairs. The enzyme uses isovaleryl-CoA or isobutyryl-CoA and three molecules of malonyl-CoA to form phlorisovalerophenone or phlorisobutyrophenone, intermediates in the biosynthesis of the hop bitter acids (alpha- and beta-acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The pI of the enzyme was 6.1. Km values of 4 microm for isovaleryl-CoA, 10 microm for isobutyryl-CoA and 33 microm for malonyl-CoA, were found. The amino-acid sequences of two peptides, obtained by digestion of VPS, showed that the enzyme is highly homologous to plant chalcone synthases. The functional and structural relationship between VPS and other aromatic polyketide synthases is discussed.