Mechanism of horseradish peroxidase inactivation by benzhydrazide

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618 S. M. Aitken and others Figure 6 Absorption spectra of peaks (A) 1, (B) 2, (C) 4 and (D) 5 from chromatogram B in Figure 4 Table 1 k inact , K I ,IC 50 and K d values for the inactivation of HRPC by arylhydrazides K I and k inact determined as described in the Materials and methods section. Preincubation conditions for IC 50 determinations were 10 nM HRPC, 1 mM H 2 O 2 and (1–5) × 10 4 µMhydrazide in 100 mM sodium phosphate, pH 7.0, for 1 min at 25 ◦ C. IC 50 values are means from at least three experiments. MPO IC 50 values are from [10] and K d values are from [31]. -OH-BZH, -hydroxybenzhydrazide; -CH 3 -BZH, -methylbenzhydrazide; -NH 2 -BZH, -aminobenzhydrazide; -Cl-BZH, -chlorobenzhydrazide; -OH-NZH, -hydroxybenzhydrazide; -NO 2 -BZH, -nitrobenzhydrazide; NICH, nicotinic hydrazide; 2-NZH, 2-naphthoichydrazide. Inhibitor k inact (min −1 ) K I (mM) IC HRPC 50 (mM) IC MPO 50 (mM) K d (mM) K I /K d 2-NZH 0.14 + − 0.01 0.014 + − 0.006 0.0350 + − 0.0008 0.0052 + − 0.0001 2.7 3-CH 3 -BZH 0.049 + − 0.006 0.7 + − 0.2 0.58 + − 0.03 0.108 + − 0.003 6.3 BZH 0.035 + − 0.003 0.08 + − 0.03 1.00 + − 0.05 0.042 0.120 + − 0.002 0.66 3-OH-BZH 0.073 + − 0.003 0.7 + − 0.1 0.8 + − 0.2 0.120 + − 1 5.6 4-CH 3 -BZH 0.088 + − 0.004 0.45 + − 0.05 0.48 + − 0.03 0.143 + − 5 3.1 4-NH 2 -BZH 0.45 + − 0.02 1.4 + − 0.2 2.2 + − 0.4 0.002 0.23 + − 0.02 6.1 3-NH 2 -BZH 7 + − 2 60+ − 20 1.5 + − 0.1 0.25 + − 0.01 320 3-Cl-BZH 0.025 + − 0.003 1.4 + − 0.5 2.4 + − 0.1 0.27 + − 0.01 5.2 4-Cl-BZH 0.33 + − 0.01 6.0 + − 0.5 2.0 + − 0.2 0.25 0.30 + − 0.01 20 4-OH-BZH 0.41 + − 0.01 0.28 + − 0.03 0.52 + − 0.02 0.018 0.54 + − 0.02 0.52 2-OH-BZH > 10 5.7 + − 0.2 0.63 + − 0.02 54 2-Cl-BZH 0.13 + − 0.02 7 + − 2 22+ − 1 2.05+ − 0.04 3.6 2-CH 3 -BZH > 10 11 + − 2 2.76+ − 0.03 13 4-NO 2 -BZH > 10 3.9 + − 0.3 3 + − 1 0.76 2-NH 2 -BZH 0.47 + − 0.02 0.9 + − 0.1 1.1 + − 0.2 3.1 + − 0.2 0.27 INH 0.025 + − 0.002 1.4 + − 0.4 3.3 + − 0.2 3.36 + − 0.04 0.42 NICH 0.014 + − 0.003 0.6 + − 0.5 8.5 + − 0.1 4.6 + − 0.1 0.13 3-NO 2 -BZH > 10 5.9 + − 0.3 9.11 + − 0.05 0.20 2-NO 2 -BZH 0.03 + − 0.02 9 + − 10 61 + − 4 90+ − 30 0.11 1-NZH 0.46 + − 0.01 0.94 + − 0.08 0.6 + − 0.1 3-OH-2-NZH 0.05 + − 0.01 0.3 + − 0.2 0.25 + − 0.03 PHZ undergoes both extensive auto-oxidation and haem-catalysed oxidation at physiological pH [20,29]. Auto-oxidation should not impede the development of hydrazides as potential therapeutics. However, recent results have demonstrated that the therapeutic effects of INH are due to its oxidation via the superoxide pathway rather than the peroxidase pathway of KatG [13,28]. Therefore, further work on hydrazide-dependent free radical generation catalysed by different peroxidases is a necessary step in assessing the potential of these compounds as therapeutic agents. HRPC inactivation by H 2 O 2 Approx. 0.12 µmol of O 2 was produced during the consumption of 0.43 µmol of H 2 O 2 (approx. 300 molar equivalents) by 1.5 nmol c○ 2003 Biochemical Society
Inhibition of horseradish peroxidase by arylhydrazides 619 Scheme 1 Mechanism of HPRC-catalysed O 2 consumption in BZH solutions HRPC(Fe III )oxidizestheBZH − anion to give HRPC(Fe II )andtheBZH • radical. O 2 reacts with the reduced peroxidase to give HRPC(Fe II O 2 )(Compound III). Using BZH − as a donor, reductive cleavage of the O–O bond of compound III yields Compound I, which is reduced by two equivalents of BZH − in two single-electron steps to yield BZH • and HRPC(Fe III ). The BZH • radicals react with O 2 to give O −• 2 and/or a peroxy radical. Compound III can also be formed by the reaction of O −• 2 with HRPC(Fe III ). Definitions of Compounds I and II are given in the Discussion. of HRPC over 30 min. This observation is consistent with previous reports that HRPC is able to consume H 2 O 2 by a catalytic mechanism (2H 2 O 2 → O 2 + 2H 2 O) via the following reactions [34,35]: HRPC(Fe III ) + H 2 O 2 → Compound I + H 2 O (4) Compound I + H 2 O 2 → HRPC(Fe III ) + O 2 + H 2 O (5) where Compound I is the two-electron-oxidized intermediate formed when 1 molar equivalent of H 2 O 2 reacts with HRPC(Fe III ). The rate-limiting step in O 2 production is reaction (5), which has arateconstant of 5 × 10 2 M −1 · s −1 [34]. Compound II (the species formed on one-electron reduction of Compound I) can also react with H 2 O 2 (with a rate constant of 2.1 M −1 · s −1 [34]) to give Compound III [36,37]: Compound II + H 2 O 2 → HRPC(Fe II O 2 ) + H 2 O (6) As discussed above, HRPC(Fe II O 2 ) can reversibly eliminate O 2 or O −• 2 (Scheme 1 [30]). Additionally, HRPC(Fe II O 2 ) decays irreversibly to p-670 (Figures 3A and 4B [27]), which contains abiliverdin-like (open-ring porphyrin lacking iron) prosthetic group (m/z 583). Formation of both Compound III (maximal by approx. 3.5 min) and p-670 (maximal by approx. 30 min) were observed here during the turnover of approx. 300 molar equivalents of H 2 O 2 by HRPC (Figure 4C). HRPC inactivation by BZH/H 2 O 2 Inactivation of HRPC by BZH/H 2 O 2 probably involves covalent modification of the enzyme since removal of low-molecularmass species by gel filtration did not restore activity (results not shown). The correlation between HRPC concentration and the rate of enzyme inactivation (Figure 3D) suggests that release and rebinding of an activated BZH species occurs prior to inactivation, as expected for a metabolically activated inhibitor. This is supported by the observations that inactivation is timedependent (Figures 3A and 3B) and that equimolar guaiacol provided complete protection against inactivation (Figure 3C). The isolation of hydroxyl-haem (m/z 632) and benzoyl-haem (m/z 720) adducts provides further evidence for covalent modification of HRPC by BZH/H 2 O 2 .Nonetheless, native haem was the predominant species isolated from BZH/H 2 O 2 -inactivated HRPC (Figure 5). The yield of hydroxyl-protoporphyrin IX haem was dramatically higher in HRPC samples treated with PHZ/H 2 O 2 (results not shown) than with BZH/H 2 O 2 (Figure 5). It is possible that HRPC inactivation by BZH/H 2 O 2 is due predominantly to modification of the polypeptide rather than the haem, and a phenyl-protein adduct was detected (but not localized) in PHZ/H 2 O 2 -inactivated HRPC [2]. Modification of protein residues around the active site by benzoyl adduct formation would result in diminished access to reducing substrates but not to H 2 O 2 , thus accelerating the formation of Compound III and p-670, as observed in the presence of BZH/H 2 O 2 (Figure 4C). Based on their masses, absorption spectra (Figure 6), and the reported haem shielding by the polypeptide that directs reactants to the δ-meso-position [2,38], the modified haems extracted from BZH/H 2 O 2 -treated HRPC (Scheme 2, compounds 9 and 10) are assumed to be δ-meso-benzoyl-haem (m/z 720) and 8-hydroxymethyl-haem (m/z 632). The growth and decay of 825-nm absorption during the inactivation of HRPC by phenylethylhydrazine and H 2 O 2 was attributed to the transient formation of an isoporphyrin cation following attack of the phenylethyl radical on the haem of Compound II [3]. The detection of transient 820-nm absorption (Figure 4C) indicates that a similar isoporphyrin cation intermediate (p-820) leads to benzoyl-haem formation (Scheme 2, compound 10) in the HRPC/BZH/H 2 O 2 reaction. This transient cation is formed from compound 8 on donation of the unpaired electron to the haem iron (Scheme 2). No transient 820-nm absorption was detected in the HRPC/H 2 O 2 reaction (Figure 4B), confirming that p-820 formation is hydrazidedependent. Formation of hydroxyl-haem (compound 9) is also shown in Scheme 2 and is based on the mechanism proposed for HRPC inactivation by PHZ/H 2 O 2 [2]. Abstraction of a hydrogen atom from the haem (compound 6) by the benzoyl radical (compound 5) results in a carbon-centred radical (compound 7) that is oxidized by the Fe IV = O centre. Subsequent addition of water to the cation leads to the formation of compound 9. Water rather than O 2 was observed to be the source of the hydroxyl group in the HRPC/ PHZ/H 2 O 2 reaction [2]. Two mechanisms for benzoyl radical generation are considered (Scheme 2). Following mechanism 1, proposed for INH activation [39], one-electron oxidation of BZH by Compound I (or Compound II) yields the BZH • radical (compound 2) which eliminates NH = NH (di-imide) to produce the benzoyl radical (compound 5). In the second mechanism, based on that proposed for PHZ activation [2], BZH is oxidized in three single-electron steps by Compounds I/II to the diazene radical (compound 4). N 2 release from compound 4 gives the benzoyl radical (compound 5), which covalently modifies the peroxidase. Both mechanisms could be operative in HRPC inactivation by BZH/H 2 O 2 for the following reasons. (i) Oxidation of BZH (compound 1) would compete with oxidation of the diazene (compound 3) or inactivation by the free benzoyl radical (compound 5) so that the efficiency of inactivation would decrease at higher BZH/H 2 O 2 ratios as observed in Figure 3(E). (ii) The release and oxidation of BZH • (compound 2) or the benzoyl radical (compound 5) from the active site of HRPC, or the direct formation of compound 5 from compound 2 would result in O 2 consumption, which was observed during the reaction of HRPC with BZH/H 2 O 2 (results not c○ 2003 Biochemical Society
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Mechanism of horseradish peroxidase inactivation by benzhydrazide

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