Haloperoxidase activities

Kataoka M, K Honda, S Shimizu (2000) 3,4-Dihydrocoumarin hydrolase with haloperoxidase activity from Acinetobacter calcoaceticus. Eur J Biochem 267 3-10. 

Mehrtens G (1994) Haloperoxidase Activities in Arctic Macroalgae. Polar Biol 14 351... 

Sheng D, Gold MH (1997) Haloperoxidase Activity of Manganese Peroxidase from Phanerochaete chrysosporium. Arch Biochem Biophys 345 126... 

The standard assay for haloperoxidase activity is the halogenation of monochloro-dimedone (mcd) (2-chloro-5,5-dimethyl-l,3-dimedone) using dihydrogen peroxide as the oxidant of the halide (Figure 4) [48],... 

Figure 4 The monochlorodimedone (mcd) reaction for measurement of haloperoxidase activity.

Bromoperoxidase activity is expressed as micromoles of mcd brominated per minute per milligram of enzyme (U/mg). The early work on V-BrPO employed the oxidation of iodide by dihydrogen peroxide [1], forming triiodide (Ir), which was followed spectrophotometrically at 353 nm ( = 26,400 M 1cm 1). However, this reaction is less desirable for quantitation of haloperoxidase activity because of competing side reactions, such as the nonenzymatic oxidation of iodide by dihydrogen peroxide and reduction of triiodide by dihydrogen peroxide (discussed later). The specific activity of mcd bromination for V-BrPO isolated from A. nodosum is 170 U/mg (at pH 6.5, 2 mM H202, 0.1 M Br , 50 nM mcd,... 

Recently the amino acid sequence of vanadium chloroperoxidase was determined to have similar stretches with three families of acid phosphatases, which were previously considered unrelated [72], This sequence raises questions about the phosphatase activity of apo-V-ClPO and whether the acid phosphatases can coordinate vanadate and carry out peroxidative halogenation chemistry. In fact, apo-V-C1PO does have phosphatase activity, catalyzing the hydrolysis of/i-nitrophe-nol phosphate (p-NPP). In addition, /i-NPP displaces vanadate from V-CIPO. At this point, the haloperoxidase activity of the acid phosphatases containing coordinated vanadium(V) has not been reported. 

These oxidations proceed without a catalyst if the concentration of the reagents in the aqueous phase is above 3 M in pH<3, but the reaction rate falls rapidly to zero (particularly in oxychlorination and oxybromination) at lower concentrations or at higher pH. The standard assay for haloperoxidase activity is the chlorination/bromination of chlorodi-medone (equation 49)368. 

Haloperoxidase activity is usually determined spectrophotometri-cally by the bromination or chlorination of 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochlorodimedone MCD) using hydrogen peroxide as the oxidant of the halide (28) (Figure 2). This assay is convenient because of the large change in extinction coefficients between the enolate and the product. However, its use is constrained by the need for conditions that stabilize the enolate. In the early work on V-BrPO, the... 

The specific chloroperoxidase, bromoperoxidase, and iodoperoxi-dase activities differ substantially and depend on pH and the concentrations of halide and hydrogen peroxide (2). In general, the specific activity for halide oxidation increases in the order of chloride, bromide, and iodide. The pH for maximum specific haloperoxidase activity generally decreases in the order of iodide, bromide, and chloride, but direct comparisons are difficult because the pH maximum can be shifted over several pH units by varying the ratio of halide to hydrogen peroxide and because both halide and hydrogen peroxide can inhibit the enzyme under certain conditions. 

Figure 3. Bromination of TMB is an alternative assay for haloperoxidase activity.

The pseudohalide azide inhibits VCIPO. The first stmcturally characterised VHPO had in fact been crystallised in its azide-inhibited form. Inhibition has also been noted with hydroxylamine and hydrazine. Further, structural analogues of vanadate, such as [ALF4] and phosphate, are potent inhibitors. In turn, vanadate inhibits many phosphatases (and other phosphate-metabolising enzymes). On the other hand, apo-VHPOs can exhibit some phosphatase activity, and vanadate-inhibited phosphatases show some haloperoxidase activity. These phenomena will be discussed in Section 5.2.1. 

Haloperoxidase Activity of Vanadate-inhibited Phosphatases Phosphatase Activity of Apo-haloperoxidases... 

For the same systems haloperoxidase activity has been reported with H2O2 / O2 as oxidant and HC1 / HBr as halogen source [22]. In this way oxychlorination / oxybromination of benzene, toluene, phenol, aniline, anisolc and resorcinol could be achieved. 

Vanadate and Phosphatases. The structural similarity between vanadate and phosphate is impressively demonstrated by the fact that apo-VHPOs can exhibit some phosphatase activity, whereas certain vanadate-inhibited phosphatases exert haloperoxidase activity, a fact which roots in homologies of the active site protein pockets of both classes of enzymes, and the structural analogy of the active centers (the histidine-coordinated vanadate) in the VHPOs and the phosphatases (17) cf Fig. 2. Vanadate-inhibited phosphatases for which peroxidase activity has been reported are of bacterial Shigella flexneri, Salmonella enterica (18)) and fungal origin (ph3rtases from Aspergillus (19)). 

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