Chloroperoxidase-catalyzed halogenation of antipyrine, a drug substrate of liver microsomal cytochrome P-450

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 210, No. 1, August, pp. 167-178, 1981

Chloroperoxidase-Catalyzed Halogenation of Antipyrine, a Drug Substrate of Liver Microsomal Cytochrome f-450’

PATRICIA L. ASHLEY AND BRENDA WALKER GRIFFIN’

Biochemistry Department, The University of Texas Health Science Center at Dallas, 5S2S Harry Hines Boulevard, Dallas, Texas 752.Q?5

Received February 11, 1981

Chloroperoxidase catalyzes halogenation of antipyrine at the 4-position with stoichiometric HzOz, in the presence of excess KC1 or KBr; the pH optima of these reactions, near pH 4.0, are characteristic of other halogenation activities of this enzyme. In the presence of KC1 or KBr, antipyrine was an effective inhibitor of HzOz decomposition catalyzed by chloroperoxidase. Under similar experimental conditions, I-halogenation of antipyrine also occurred in the absence of chloroperoxidase with stoichiometric NaOCl, or enzymatically with horseradish peroxidase in the presence of Hz02 and excess KBr (but not KCl). As observed previously for chloroperoxidase, horseradish peroxidase catalyzed oxidation of Br- by Hz02 to Brz, readily detected in the presence of excess Br- by the intense uv absorbance of Br;. These nonenzymatic and enzymatic halogenating systems could also effect N-demethylation of antipyrine, with complete release of formaldehyde requiring a severalfold molar excess of NaOCl or HzOz, respectively. These results and data obtained with 4-bromoantipyrine indicated that the methyl group is cleaved subsequent to halogenation. Since the halide anion was absolutely required for the enzymatic N-demethylation reactions, it appears that the enzymatically generated halogenating species is also responsible for N-demethylation, which is much less favorable than halogenation of antipyrine. These results parallel qualitatively the relative extents of I-hydroxylation and N-demethylation of antipyrine catalyzed by liver microsomal eytochrome P-450 in vivo and in vitro and provide evidence for similar functions of chloroperoxidase and cytochrome P-450 as catalysts of: (1) dehydrogenation reactions and (2) insertion of an electonegative atom (Cl or 0) into an organic compound,

The mechanism by which cytochrome P- 450 activates molecular oxygen for the monooxygenation of many electron donor substrates, resulting in insertion of an oxygen atom into a C-H bond, is the subject of increasing research interest (1, 2). The proposal that these reactions involve a species of cytochrome P-450 analogous to Compound I of horseradish peroxidase (HRP),3 which may be formed with either O2 and NADPH (requiring NADPH-cyto-

1 Supported by NIH Grant AM 19027 and Grant I- 601 of the Robert A. Welch Foundation, to B.W.G.

‘To whom reprint requests and correspondence should be sent.

’ Abbreviations used: CPO, chloroperoxidase; HRP, horseradish peroxidase; EPR, electron paramagnetic resonance; tle, thin-layer chromatography.

chrome P-450 reductase) or various organic hydroperoxides (3, 4), has not been supported by recent work published from this laboratory and others (5, 6). The hydroperoxide-supported N-demethylation of several cytochrome P-450 substrates has been shown to involve radical species of these electron donors, whether catalyzed by purified liver microsomal cytochrome P-450 (6) or other hemeprotein peroxidases (7). The chemistry of this reaction-a two-electron dehydrogenation of the substrate, followed by hydrolysis of the iminium cation intermediate-has strongly implicated HzO, and not the oxidant, as the source of oxygen atom of formaldehyde (6, 7). These results have suggested the possibility that the 0.J

167 0003-9861/81/090167-12$02.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

168 ASHLEY AND GRIFFIN

NADPH-supported monooxygenation of N-methyl compounds catalyzed by cytochrome P-450 might also involve one-electron oxidation of these substrates, but several experimental problems have precluded a definitive test of this hypothesis (6, 7).

Therefore, we have taken another ap- proach to model-system studies by using a more appropriate hemeprotein catalyst and electron donor substrate. Chloroper- oxidase (CPO) utilizes H202 or certain other peroxidic agents to oxidize halides (except F-) to electrophilic species which insert into @keto carboxylic acids or car- bonyls, and other compounds (8). Signifi- cantly, this hemeprotein possesses most of the unique physical properties of the cytochrome P-450 class of enzymes, in- cluding the characteristic absorbance maximum of the ferrous hemeprotein-carbon monoxide complex near 450 nm (8,9). The functional significance of these re- markably similar structural features has not yet been elucidated, but the implica- tion is that halogenation and hydroxylation reactions catalyzed by these respective hemeproteins may be similar in certain mechanistic aspects. In a study designed to provide a test of this hypothesis, we have first investigated whether electron donor substrates of cytochrome P-450 may also undergo halogenation catalyzed by CPO. Although certain cyto- chromes P-450, i.e., from adrenal mito- chondria and from camphor-grown P. putida (lo), exhibit rather stringent specificity for the electron donor substrate, the membrane-bound liver microsomal cytochrome P-450 can catalyze the oxidation of many classes of substrates, due in part to the existence of several inducible forms of this hemeprotein (2,11). Little is known about the degree of overlap of substrate specificity of liver microsomal cytochrome P-450 and CPO. Although it was recently reported that CPO catalyzes the N-demethylation of N,N-dimethylaniline (12), those experimental results did not elimi- nate the possibility that this CPO-catalyzed reaction also occurs by a radical dehydrogenation process, rather than enzyme-mediated insertion of oxygen from the oxidant (7, 13). In the present study,

we have demonstrated that antipyrine, a drug which is metabolized in vitro by liver microsomal fractions (14) and in vivo (15) to several products, can undergo analogous halogenation/oxidation reactions catalyzed by CPO.

MATERIALS AND METHODS

The CPO used in this study was a preparation from Caldatiomycesfumago supplied by Sigma. By several criteria, it appears to be the same protein which has been characterized extensively in Hager’s laboratory. The specific activity, measured by the standard assay of monochlorodimedone chlorination (16), was 7.75 X lo* pmol substrate consumed/min/mg protein. Although this value is less than the specific activity reported for a crystalline preparation of the enzyme (1.6 X 10a~mol/min/mg (16)), it appears to be greater than the specific activity of preparations employed in several earlier studies, as determined from actual data contained in those publications (17,lS). The pH dependence of enzymatic chlorination of monochlorodimedone with either H20z and KC1 or NaC102 was very similar to that previously reported (18), with a sharp optimum near pH 2.8. In addition, the relative specific activities of this preparation of CPO for bromination of monochlorodimedone and for oxidation of I- to I2 agreed with those previously reported (17). Spectral properties of the hemeprotein were also examined in order to confirm their similarity to those of both CPO (8, 9) and cytochrome P-450 (1, 2). The Soret absorbance of the ferric hemeprotein, in the absence of added halide anion, and of the carbon monoxide complex of the ferrous hemeprotein, both measured at pH 3.0, occurred at 395 and 444 nm, respectively. For CPO and bacterial cytochrome P- 450, the positions of these absorbance maxima of the two hemeprotein forms have been established to be 396 and 443 nm and 392 and 446 nm, respectively (1, 9). The reported effects of high concentrations of KBr and KC1 on the Soret absorbance of the ferric form of CPO were also reproduced with this preparation, namely KBr produced a type I spectral change and KC1 caused a Type II spectral change (9). Consistent with previous observations (9), we noted that CPO was converted near neutral pH to a form having the spectral characteristics of the inactive cytochrome P-450 (1). Based on these data, it is concluded that the CPO used in this study is the same, or a very similar, hemeprotein as that isolated from the same source, and previously characterized (8).

The HRP employed in this study was purchased from Sigma (Type VI, salt-free powder) and was di- alyzed extensively against distilled water before use. Hydrogen peroxide, 30% AR Grade without stabiliz- ers, was a product of Mallinckrodt; concentrations of stock solutions of this reagent were determined

CHLOROPEROXIDASE-CATALYZED HALOGENATION OF ANTIPYRINE 169

spectrophotometrically at 240 nm, at which wavelength t = 0.0394 mM-' cm-’ (19). Antipyrine was obtained from Aldrich, and 4-bromoantipyrine from the ABC Library of Rare Chemicals (Aldrich). All other chemicals were the highest quality commer- cially available.

Absorbance measurements were made with a Beckman Model 25 uv-visible spectrophotometer. A Clark-type Oz electrode was utilized to monitor Oz levels continuously in solutions contained in a water- jacketed cell designed for such measurements; for calibration purposes, the decomposition of Hz02 catalyzed by catalase was employed. Plastic tic plates, 20 X 20 cm, coated with silica gel 60 (0.2 mm thick- ness), containing a fluorescent indicator, were supplied by E. Merck. In initial experiments, two different solvent systems were investigated for development of the tic plates: Sl containing CHCla, methyl-t-butyl ether, and methanol (2:2:1) and S2, containing hexane, ethyl acetate, and glacial acetic acid (15:50:5). Since system 52 gave better resolution of antipyrine and 4-bromoantipyrine, it was employed in subsequent experiments. In a typical ex- periment, the desired amount of Hz02 or NaOCl was added in several aliquots to a reaction mixture containing 10 mM antipyrine and other required com- ponents in a buffered solution; the reaction mixtures for specific experiments are described in detail in Table I. The course of the reaction was monitored in the uv region on diluted samples of the reaction mixture. Then 2 ml of the reaction mixture was extracted with two 2-ml portions of solvent, evaporated to dry- ness under a stream of dry Nz, redissolved in 0.1 ml of solvent, and a 2-to 5J sample loaded on the tic plate, along with equivalent amounts of antipyrine and 4-bromoantipyrine as standards. The solvent commonly employed for the extraction was CHCl,; however, the same results were obtained when the reaction mixture was extracted with ethyl acetate or dichloroethane. Formaldehyde was assayed by the standard Nash procedure (20), after the reaction had been quenched with 15% trichloroacetic acid and cen- trifuged to remove precipitated protein. The appropriate control experiments were performed to establish that formaldehyde was not destroyed in any of the reaction systems assayed. Concentrations of stock solutions of NaOCl were determined by oxidation of I- in the presence of excess II; the concentration of I; was computed from the absorbance of 353 nm, for which c is 2.6 X lo4 M-' cm-i (21), and free Iz was negligible under the experimental conditions employed. NMR spectra were recorded with a Perkin-Elmer spectrometer operating at 90 MHz.

RESULTS

In the presence of CPO and excess KBr, the uv spectrum of antipyrine was modi-

TABLE I T~~tlc IDENTIFICATIONOF HALOGENATED

PRODWTSOFANTIPYRINEPRODUCEDWITH NaOCl ORENZYMATICALLYWITHCPO

Reaction” Rf Values

1 0.16*, 0.34 2 0.16*, 0.33 3 0.16*, 0.34

Antipyrine standard 0.16 4-Bromoantipyrine standard 0.35

“Composition of the reaction mixtures: 1, 0.1 M potassium phosphate, pH 3.0, 0.1 M KCl, 10 mM antipyrine, ‘76 nM CPO (2 aliquots), and 8 mM Hz02 (4 aliquots); 2, 0.1 M potassium phosphate, pH 3.0, 10 mM antipyrine, and 8 mM NaOCl (4 aliquots); 3, 0.1 M potassium acetate, pH 4.0, 0.1 M KBr, 10 mM antipyrine, ‘76 nM CPO (2 aliquots), and 8 mM Hz02 (2 aliquots).

*Weak spot (unreacted antipyrine).

fied as shown in Fig. IA by the addition of increasing amounts of HzOz, to 1 molar eq relative to the antipyrine concentration. A good isosbestic point was main- tained throughout this titration, indicating that antipyrine was converted to a single stable product by stoichiometric reaction with HzOz. All control experiments were completely negative: CPO and HzOz, at the concentrations employed in Fig. lA, had no measurable absorbance in this region, and the reaction did not occur if KBr, CPO, or HzOz was omitted. When KC1 was substituted for KBr, antipyrine could be similarly titrated in the presence of CPO with 1 molar eq of Hz02 (Fig. lB), resulting in a final absorbance spectrum very similar to that produced with KBr (Fig. 1A). In the presence of 0.1 M halide anion, the pH optima for both reactions were very broad, approximately 3.0 to 4.5 for the Br-- dependent reaction and 3.0 to 4.0 for the Cl--requiring reaction. The pH optima for other halogenation reactions catalyzed by CPO have been shown to fall within this range and to depend upon the actual halide concentration (18, 22). In the absence of CPO, addition of stoichiometric NaOCl to antipyrine in buffered solutions produced an absorbance change (Fig. 1C) identical to that observed during the Cl--dependent enzymatic reaction (Fig. 1B); over the pH


225 275 325

0.8

E 5 0.6

E g 0.4

:

0.2

225 275 325

D RI

225 275 325

WAVELENGTH, nm

FIG. 1. The uv absorbance spectral changes observed during the stoichiometric halogenation of antipyrine with NaOCl or enzymatically with CPO. A and B: The sample cuvet contained 100 PM antipyrine, with absorbance maxima at 243 and 256 nm (-), and 10 nM CPO (no measurable absorbance) in 0.1 M sodium acetate buffer, pH 4.0, with 0.1 M KBr (A) or 0.1 M sodium phosphate buffer, pH 3.0, with 0.1 M KC1 (B). The series of absorbance spectra were recorded approximately 4 min after each 20 PM addition of HrOs, to a final concentration of 100 PM (- - -). C: To 100 PM antipyrine in 0.1 M sodium phosphate, pH 3.0, in the sample cuvet (L) were added successive aliquots of 29,29,29, and 10 PM NaOCl, and the absorbance spectrum scanned approximately 2 min after each addition. D: 100 pM I-bromoantipyrine in 0.1 M sodium acetate, pH 4.0.

range 3.0 to 7.0 (which was the highest pH examined), this nonenzymatic reaction was very fast.

It has been previously reported that antipyrine can be halogenated at the ~-PO- sition in high yields by two different chemical halogenating agents, Clz (23) and N-bromosuccinimide (24). The uv spectrum of an authentic sample of 4-bromoantipyrine (Fig. 1D) was identical to that of the product of CPO-catalyzed bromination of antipyrine (Fig. 1A). In each of the three halogenating systems described, a single product was formed from anti-

pyrine with stoichiometric oxidant or less, and this product exhibited the same Rf value as the 4-bromoantipyrine standard, Table I. The NMR spectra of the three isolated products could not be distin- guished from that of the 4-bromoantipyrine standard, Fig. 2; three distinct groups of protons were observed, with chemical shifts and intensities expected for this chemical structure (cf. Fig. 7): five aromatic protons near 6 = 7.3 and two groups of methyl protons at 6 = 3.0 and 6 = 2.2. These data conclusively identify 4-bromoantipyrine as the product of the CPO- catalyzed reaction of antipyrine in the presence of KBr. Although an authentic sample of 4-chloroantipyrine could not be obtained, this product is identified by the conditions of its formation and by the similarity of its uv, NMR, and chromato- graphic properties to those of 4-bromoantipyrine. The halogen substituent is sufficiently far removed from the aromatic ring and also from the other protons (cf. Fig. 7) that the uv and NMR spectra are affected only slightly or not at all, respectively, by the identity of the halogen.

With excess HzOz in the two enzymatic systems or excess NaOCl in the absence of CPO, further changes in the uv spectrum of the respective 4-haloantipyrine

TMS-

‘4

smLl-!L IO 8 6 4 2

d. ppm

FIG. 2. Comparison of the NMR spectra of I-bromoantipyrine (A) and the isolated product of enzymatic chlorination of antipyrine (B). Both samples were dissolved in CDCls at concentrations of 0.15 M, with tetramethylsilane (TMS) added as the reference standard. Instrument settings were 10 ppm sweep range; 450 s sweep time; filter, 2.0; H1 level, 9; and sensitivity, 1.0.


were observed, consistent with the ap- pearance of two weak spots on the tic plates with Rf values of 0.66 and 0.33. The possibility that these products were as- sociated with N-demethylation, and subsequent reactions, of the 4-haloantipyrine was suggested by the following results. The production of formaldehyde from antipyrine in these three halogenating systems could be observed only when an excess of HzOz, or NaOCl, relative to antipyrine was employed, indicating that N-demethylation occurs @er halogenation of the substrate. However, the addition of an equivalent amount of HzOz to I-bromoantipyrine in the enzymatic brominating system prduced approximately one-third the expected amount of formaldehyde (Fig. 3A), based on a stoi- chiometry of one formaldehyde produced per Hz02 consumed (7); the subsequent addition of 2 molar eq of HzOz yielded the theoretical maximal value of formaldehyde from this compound. As shown in Fig. 3A, all controls, in particular the ex- periment in which KBr was omitted, were negative. Qualitatively similar results were obtained when increasing amounts of NaOCl were added to either of the 4-haloantipyrines in the absence of CPO under identical experimental conditions (Fig. 3B). However, the yield of formaldehyde

’ ’ -‘. ’ a 0 -IL ’ 8 fi - 0 2 4 6 0 2 4 6 0 2 4 TIME. MIN t t t

A 1.0 2.0 3.0 H,O, s &y,‘b”,‘c

with 1 molar eq of NaOCl was less than that observed in Fig. 3A with HzOz, and consequently, more NaOCl, i.e., 4 molar eq, were required for complete oxidative cleavage of the N-methyl group. The data of Figs. 3A and B indicate, and the appropriate control experiments demonstrated, that formaldehyde was completely stable under these experimental conditions. Our attempts to isolate and identify the desmethyl product, a secondary amine, pre- sumed to be formed along with formaldehyde, by use of excess oxidant in these several systems were unsuccessful. Indeed, the requirement for a severalfold excess of oxidant for complete release of formaldehyde suggests that this amine is readily oxidized under these experimental conditions. This interpretation is quite consistent with the known reaction of secondary amines with NaOCl to form N- chloramines, which are also highly reac- tive species (25). Finally, we note that the chemical oxidation of structural analogs of antipyrine, under acidic conditions, has been extensively studied (26-28); several products have been identified, resulting from both N-demethylation of the pyrazolone nitrogen and oxidative attack at various positions of this ring (26-28). The two unidentified products detected by tic when excess oxidant was utilized in these

0 30 3

% I 0 31 ‘IME. MIN

t t t t B 1.0 2.0 3.0 4.0

NoOCl.molar aquiv.

FIG. 3. N-Demethylation of I-haloantipyrines catalyzed by CPO in the presence of KBr or resulting from reaction with NaOCl. Both reactions were carried out in 0.1 M sodium acetate buffer, pH 4.0, with 0.4 mM of the respective I-haloantipyrine, A (Br) and B (Cl), and aliquots were withdrawn for assay of formaldehyde (H&O) at the indicated times. For A, three additions each of 0.4 mM HrOr and 6’7 nM CPO were made at zero time(s): (0), + 0.1 M KBr; (O), control without KBr. Controls without CPO or HzO, were similarly negative. For B, the procedure was similar except that no halide was present, and four separate aliquots of 0.4 mM NaOCl were added at zero time(s), as indicated.


various systems probably arise from further oxidation of the desmethyl halogenated product.

It has been previously reported that CPO has a significant catalatic activity, which is stimulated by Br- or Cl- (22,29). In the presence of KBr, antipyrine was a potent inhibitor of the extent of O2 formation from H202 catalyzed by CPO, as shown in Fig. 4. The dependence of the relative inhibition by antipyrine on the H202 concentration suggests that antipyrine competes effectively with HzOz as an electron donor in this reaction system. In the absence of added halide, the CPO-catalyzed evolution of 02 from HzOz occurred at a slower rate, consistent with previous observations, and was not inhibited by any concentration of antipyrine up to 10 I’nM;

indeed, this concentration of antipyrine slightly increased the amount of Oz produced. The control experiments demonstrated that under all conditions, i.e., with or without the halide, antipyrine, or both, the decomposition of Hz02 absolutely required the enzyme. Consistent with the evidence presented above for enzymatic N- demethylation of 4-bromoantipyrine, this compound also inhibited the catalatic activity of CPO in the presence of KBr, but less effectively than antipyrine, as shown

120

!

\

90 5 “\, 3 d 60 A-i

ANTIPYRINE .PM

FIG. 4. Inhibitory effect of antipyrine on bromide- stimulated Oz evolution from HzOz catalyzed by CPO. The reaction mixtures, containing 0.1 M potassium acetate buffer, pH 4.0,O.l M KBr, and the stated concentration of antipyrine, were equilibrated at 25°C in the Oz electrode cell prior to adding 6’7 nM CPO and HzO, 0.2 mM (0) or 0.4 m?d (0), to start the reaction. The rates were first order in HzOz.

-0 0.3 0.6 0.9 1.2 1.5

SUBSTRATE, mM

FIG. 5. Comparison of the inhibition by antipyrine and I-bromoantipyrine of bromide-stimulated decomposition of HzOz catalyzed by CPO. The experimental conditions were similar to those described in the Fig. 4 legend, with antipyrine (0) or 4-bromoantipyrine (0) present at the specified concentration.

in Fig. 5. The experiments of Figs. 3A and 5 were carried out under identical conditions in order to estimate the efficiency of Hz02 utilization for oxidation of 4-bromoantipyrine. The data in Fig. 5 show that 0.4 mM 4-bromoantipyrine decreased the 02 production from approximately 140 to 30 PM, corresponding to 220 I.LM HzOz (55 % ) utilized for reaction with the organic substrate. Of this fraction of the total H202, approximately 57% (0.126 mM) was accounted for by formaldehyde production (Fig. 3A). The remaining 43% of the HzOz which reacted with I-bromoantipyrine was probably consumed by the amine product of N-demethylation, as discussed earlier. Although these estimates of the amount of H202 utilized for several distinct reactions during the Br--dependent CPO-catalyzed oxidation of antipyrine are subject to some error, they do provide evidence for a very facile reaction of desmethyl4-bromoantipyrine under these experimental conditions. The effects of antipyrine and 4-chloroantipyrine (formed by reaction of antipyrine with 1 molar eq of NaOCl) on the Cl-stimulated decomposition of HzOz catalyzed by CPO were qualitatively similar to the data in Fig. 5, except that both compounds were somewhat more effective inhibitors of Oz evolution in this reaction system. We have observed that monochlorodimedone and other compounds known


to be halogenated by CPO similarly inhibit the catalatic activity of this enzyme, but only in the presence of Cl- or Br-. It appears that, because HzOz is such an excel- lent electron donor substrate for CPO in the presence of these halide anions, measurable enzymatic halogenation of an organic compound must correlate with its ability to inhibit the catalatic reaction.

The preceding discussion has illustrated the difficulties of measuring the true halogenation activities of CPO: (i) HzOz decomposition is a competing reaction which is more or less important depending on the substrate and the experimental conditions and (ii) in the case of antipyrine, N-demethylation of the halogenated product occurs with excess HzOz. As an estimate of the lower limit of the antipyrine halogenation activity of CPO, the rate of the enzymatic reaction, with equivalent substrate and HzOz concentrations, was measured from the difference in extinction coefficients of the reactant and product at 243 nm (cf. Fig. 1A). Under the experimental conditions of Fig. lA, with a CPO concentration (5 nM) which gave a linear initial rate, the activities with Br- and Cl- were identical, 54 pmol/min/mg protein. This value is less than 10% of the value measured for chlorination of monochlorodimedone under optimal experimental conditions with this preparation of CPO. However, we note that the standard assay of the monochlorodimedone chlorination activity of CPO calls for (16) a 20-fold excess of H202 over the organic substrate, conditions under which the rate of enzymatic decomposition of H202 is substantial. Also, the use of higher, but equivalent, concentrations of antipyrine and HzOz to determine a better value of the antipyrine halogenation activity was precluded by the very large extinction coefficients of the substrate and the halogenated product (cf. Fig. 1A). For these reasons, a quantitative comparison of these distinct halogenation activities of CPO is not possible.

It was observed that HRP, at a lo- to 20-fold higher concentration, could catalyze identically the same spectral change with HzOz and excess KBr, under the ex-

perimental conditions described in Fig. 1A. To our knowledge, HRP has not been previously reported to catalyze bromination reactions (30). The HRP-catalyzed reaction absolutely required the presence of KBr, but did not occur if KC1 was substituted for KBr. In the absence of antipyrine, the addition of CPO or HRP to limiting H202 and excess KBr resulted in formation of a species absorbing at 266 nm (Fig. 6), which was previously observed with CPO (31). There is substantial chemical evidence that this species is Bri, analogous to I;, both of which are formed by oxidation of the halide anion to the halogen, which, in the presence of a large excess of the halide anion, forms the triha- lide anion in a freely reversible reaction (32). In the previous study of CPO-catalyzed generation of this species (31), control experiments established the formation of this same species, in the presence of excess Br-, by several independent means of Brz generation; also the extinc-

230 270 310 350

WAVELENGTH (nm)

FIG. 6. Absorbance spectrum resulting from oxidation of excess Br- with limiting oxidant, enzymatically with HzO, or nonenzymatically with NaOCl. Both cuvets contained 0.1 M potassium acetate buffer, pH 4.0, containing 0.1 M KBr; 60 nM CPO (-) or 286 nM HRP (-.-) was added to the sample cuvet subsequent to the addition of 100 pM HzOz; (- - -), 26.3 PM NaOCl was added to the buffer with KBr in the sample cuvet. No absorbance developed if enzyme was omitted in the HzOz-dependent reactions or if KBr was replaced by KC1 in the three reaction systems.


tion coefficient of Br; was determined to be 2.71 X lo4 M-l cm-’ (31), which is almost identical to that of 1, in the uv region (cf. Materials and Methods) (21). As shown in Fig. 6, the 266-nm absorbing species could also be formed by adding NaOCl to KBr; however, with NaOCl and the two enzymatic systems, maximal formation of the species required a large excess of the bromide anion. NaOCl is known (33) to rapidly oxidize Br- in acidic solutions to the highly unstable HOBr, which will oxidize Br- if available. Finally, the uv absorbance maxima of various oxy-bromine species which are known clearly demonstrate that the species of Fig. 6 is not OBr- (331 nm), BrOB (290 nm), or BrO; (195 nm) (32). The experimental conditions required for enzymatic generation of measurable concentrations of Br; from Br- and limiting HzOz were very similar to those required for antipyrine bromination, and CPO was more effective than HRP as a catalyst of both reactions. As a function of pH, the maximal value of the absorbance at 266 nm occurred at pH 4.0 for both hemeproteins, but Br; was not very stable at this pH. At pH 3.0, the CPO-catalyzed reaction produced less Br3, which was quite stable under these conditions. Finally, we note that the N-demethylation of antipyrine with excess HzOz, and of 4-bromoantipyrine with equivalent HzOz, could be catalyzed by HRP in the presence of KBr. However, with HRP, larger catalyst concentrations were required, and the yield of formaldehyde was considerably less than that observed with CPO, under comparable experimental conditions. Al- though HRP has been shown to be an ex- cellent catalyst of halide-independent N- demethylation of various compounds (7, 13), under no experimental conditions examined could HRP catalyze N-demethylation of antipyrine or I-bromoantipyrine in the absence of KBr.

DISCUSSION

The results of this study have established that, in the presence of HzOz and either Br- or Cl-, CPO catalyzes the halogenation of antipyrine at the 4-position,

and subsequent N-demethylation of the product, if greater than 1 molar eq of H202 is supplied, according to the reaction scheme of Fig. 7. The nonenzymatic reaction between antipyrine and NaOCl produced qualitatively similar results under comparable experimental conditions. Sub- strates of CPO are characterized by an active hydrogen, alpha to a carbonyl or phenolic -OH group or on a sulfur atom, which can be replaced by a halogen atom (16-18, 31, 34). The hydrogen at the ~-PO- sition of’ antipyrine is considered to be similarly activated by both the carbonyl function and the five-membered pyrazolone ring. Liver microsomal cytochrome P-450 has been strongly implicated in the metabolism of antipyrine in viva by studies with both human subjects and laboratory animals (35, 36). The major urine metabolite of antipyrine in humans is 4-hydroxyantipyrine, followed by 3-hy- droxymethylantipyrine, and lesser amounts of the N-demethylated drug (15). In a recent study of antipyrine oxidation by microsomal fractions from human and rat liver, it was shown that the 4-hydroxylase activity was inducible in rats by beta- methasone and phenobarbital, while hydroxylation of the 3-methyl group was not (14). This result indicated that different molecular species of cytochrome P-450 are responsible for hydroxylation at these two positions of antipyrine. No data were presented on the inducibility of the antipyrine N-demethylase activity. However, it was

P 5% D “\,/C”, “* 0

32

Z-C”, - n.Elorc 92

- II CM3 x Cb

H’ c,,BWC I

n: HOC, B: cm, H*O* . KX p c: HRP. HIOI , KBr 0 “\“A

-IL-l + H&=0 -

x %

FIG. 7. Halogenation and subsequent N-demethylation of antipyrine effected by enzymatic halogenating systems containing CPO or HRP and by NaOCl. X = Br or Cl.


shown that the metabolite distribution obtained with microsomes from induced animals (14) was very similar to that previously found for urine metabolites of antipyrine in humans (15).

It is significant that antipyrine can serve as an electron donor substrate for both liver microsomal cytochrome P-450 and CPO, and, moreover, that two of the three reported modifications of this drug, in viva and in vitro, are analogous to those observed with CPO. We cannot exclude the possibility that halogenation of the 3- methyl group of 4-haloantipyrine may occur to some extent with CPO in the presence of excess HzOz, since the formaldehyde produced accounted for only one-third of the HzOz. Efforts to identify conclusively the products of the enzymatic reaction of antipyrine with excess HzOz are under way. However, the enzymatic insertion of oxygen or halogen, respectively, occurs preferentially at the ~-PO- sition for both CPO and an inducible form of liver microsomal cytochrome P-450 (14); and the N-demethylation reaction is less favorable with both catalysts. Unlike the results obtained with CPO, cytochrome P- 450 catalyzes N-demethylation of antipyrine, rather than the 4-hydroxylated product. The reason for this difference is un- certain, since it is not known whether one or more forms of the liver hemeprotein catalyze oxidation of antipyrine at these two positions. However, two important differences between the CPO- and cytochrome P-450-dependent reactions may explain this result: (i) very different catalytic activities of the two hemeproteins, and (ii) different reactions of the product in the two enzyme systems. For example, the catalytic activity of CPO for 4-halogenation of antipyrine is estimated to be 103-lo4 larger than the corresponding hydroxylation reaction catalyzed by liver microsomal cytochrome P-450. This value is based on the recently published data for microsomes (14) and a reasonable estimate of the specific content of cytochrome P-450 (5), which was not stated. We note that 4-hydroxyantipyrine occurs in urine almost exclusively as the glucuronide (15). If the rate of hydroxylation of antipyrine

is small compared to the rate of glucuron- idation of the reaction product, then N- demethylation of 4-hydroxyantipyrine will not occur because it does not accumulate. However, with CPO, halogenation of the drug at the 4-position occurs rapidly and quantitatively, making this product available for N-demethylation by any excess H202 present.

Other substrates of liver microsomal cytochrome P-450 which have been shown to undergo halogenation or oxidation catalyzed by CPO are limited to aniline de- rivatives. It has been reported that, in the presence of CPO, HzOz, and Cl- or Br-, p- chloroaniline is halogenated at both the 2-and 6-positions, and also undergoes N- oxidation to p-chloronitrosobenzene (37). Liver microsomal cytochrome P-450 catalyzes, at a very low rate, hydroxylation of aniline at the paru position (38). It has also been demonstrated that liver microsomal fractions catalyze oxidation of aniline and other primary aromatic amines to the corresponding N-hydroxy products, but further oxidation to the nitroso compounds was not observed (39). Thus, it is not clearly established that the CPO- catalyzed reactions of p-chloroaniline are strictly analogous to the cytochrome P- 450-dependent conversions of this class of compounds. It was recently reported that CPO catalyzes N-demethylation of NJ- dimethylaniline and related compounds by ethyl hydroperoxide, H202, and other hydroperoxides (12). Although the enzymatic reaction was stimulated by KC1 at low pH, it had a significant rate in the absence of added halide, well above the pH optima characteristic of CPO-catalyzed haloge- nations (18). Since CPO has been previously shown to catalyze the dehydrogenation of typical substrates of HRP under such conditions (22), we propose that this N-demethylase activity of CPO is similar to that exhibited by many hemeproteins in the presence of hydroperoxides (6,7,13) Several observations of Kedderis et al. (12)

support this idea: the formation of a vi- olet-colored product, along with formaldehyde, during oxidation of N,N-dimethylaniline and the preliminary report of an EPR signal correlating with higher con-


centrations of the unidentified colored product. We note that free radical species of aromatic amines can undergo various nonenzymatic reactions, the relative im- portance of which depends critically on the precise experimental conditions (40, 41). For radical N-demethylation reactions catalyzed by hemeproteins, we recently demonstrated that dimerization of the radical intermediates to form highly colored products is a significant competing reaction when radical generation rates are large and/or when electron acceptors are limiting (41). It is indeed possible that similar radical reactions may also occur in the cytochrome P-450~catalyzed oxidation of this class of compounds. However, aromatic amines cannot be considered the best choice of substrates for studies on the mechanism of oxygen insertion reactions catalyzed by cytochrome P-450 and by model compounds.

The results of this study have raised in- teresting questions about the mechanism of CPO-catalyzed halogenation and N-demethylation of antipyrine, and the rela- tionship of these activities to the analogous hydroxylation and N-demethylation of this drug catalyzed by liver microsomal cytochrome P-450. The data are consistent with involvement of HOC1 in the chloride- dependent reaction catalyzed by CPO, since NaOCl in acidic solution produced the 4- chlorinated product rapidly and quantitatively. The N-demethylation of antipyrine by excess NaOCl under similar conditions was also rapid, and, like the enzymatic N-demethylation reaction, produced lower yields of formaldehyde than expected. The direct detection of Brz as the Br; complex, implicates this species as the brominating agent produced in acidic solutions of HzOz, Br-, and either CPO or HRP. These results are consistent with those of Thomas, who showed that Brz was generated sufficiently fast to be an intermediate in the bromination activities of CPO (31). However, Thomas also demonstrated that enzymatic formation of Clz from KC1 and H202 was too slow to account for the measured rates of chlorination of most substrates of CPO (31). A consistent explanation of these results is the enzy-

matic generation of HOX, and subsequent chemical and ionization equilibria of this species which are highly dependent upon the particular halogen, the pH, and the concentration of the halide anion: for X = Br, in the presence of excess Br-, the equilibrium greatly favors Brg at all pH values less than approximately 5, whereas with X = Cl, Clz formation (Cl, is very unstable) is favored at the expense of HOC1 only in very acidic solutions, below pH 2.0 (33). Although it has been shown that HOC1 can effect a number of chlorination/oxidation reactions catalyzed by CPO (17, 31), some differences in kinetics and product distribution between the nonenzymatic and enzymatic reactions have been noted which were interpreted to mean that HOC1 is not the actual chlori- nating species generated with CPO (31). There is evidence that both HOC1 and Clz are produced in the myeloperoxidase-catalyzed oxidation of Cl- with HzOz (42).

One possible explanation of these ap- parently conflicting results about the identity of the CPO-produced halogenating species is suggested by the observation of N-demethylation of 4-haloantipyrine by all of the halogenating systems examined. Hydroperoxide-supported N-demethylation reactions, catalyzed by several hemeproteins, have been shown to occur by a radical dehydrogenation of the N- methyl group (6, ‘7, 13), similar to the mechanism of chemical N-demethylation reactions (43, 44). Thus, evidence for the generality of this mechanism suggests that N-demethylation of 4-haloantipyrine may occur by a similar radical pathway. Significantly, in the absence of added halide, neither CPO nor HRP could catalyze the N-demethylation of antipyrine under any experimental condition examined. This result indicates that the N-methyl group of antipyrine, because of steric or redox factors, cannot donate electrons directly to the higher oxidation states of HRP or CPO. Unlike antipyrine, most substrates halogenated by CPO also undergo CPO- catalyzed dehydrogenation at lower rates (31). Since these reactions do not require halide anion, they probably involve one- electron oxidation of the substrate, anal-


ogous to many other dehydrogenation activities common to both CPO and HRP (22). Substrate radicals, if produced under conditions of halogenation, could initiate a radical decomposition of HOC1 to HO. and/or Cl. radicals (45). This would be expected to result quite generally in loss of chlorination specificity and also to yield relatively more dehydrogenation products. Moreover, the distribution of products resulting from such a radical chain reaction would be expected to depend critically upon reactant concentrations and other experimental conditions (41, 46). Indeed, reported differences between CPO-catalyzed and NaOCl-dependent chlorination reactions (31) may be due to differences in steady-state levels of HOC1 which can participate in a radical chain process. However, with the appropriate substrate, having one type of hydrogen considerably more “activated” than the others (e.g., antipyrine), a radical halogenation process could be highly specific.

There is chemical evidence that both HOC1 and Brz may react by either ionic or radical pathways depending on the reactant and the experimental conditions (45,47, 48). The data of the present study do not establish which mechanism pre- dominates in the halogenation of antipyrine by HOCl, Brz, or perhaps other as-yet- unidentified enzymatic halogenating species, under the experimental conditions employed. However, the fact that antipyrine can be halogenated rather specifically at the 4-position by various chemical (23, 24) and enzymatically produced halogenating species, under quite different experimental conditions, indicates that the chemical properties of the substrate itself play an important role in determining this specificity. Although antipyrine has only a limited number of sites available for attack by a halogenating or oxidizing species, it is significant that the cytochrome P-450~catalyzed oxidation of this compound occurs on the pyrazolone ring, analogous to the CPO-catalyzed halogenation and N-demethylation reactions, and not on the aromatic ring. Aromatic ring hydroxylation is considered a characteristic activity of liver microsomal cytochrome

P-450, which has been observed with aromatic substrates having diverse chemical structures (1, 2, 11, 32). It is not clear whether the observed “specificity” of antipyrine oxidation by cytochrome P-450 reflects unusual specificity in the binding of this drug to the hemeprotein and/or chemical reactivity of the “activated oxygen” species generated in the enzymatic reaction. Studies of the mechanism of antipyrine halogenation and of CPO-catalyzed halogenation of other cytochrome P- 450 substrates are in progress, in order to resolve these questions.

ACKNOWLEDGMENT

The authors express appreciation to Dr. Edward Biehl for collaborating on the NMR experiments.

REFERENCES

1. GRIFFIN, B. W., PETERSON, J. A., AND ESTABROOK, R. W. (1979) in The Porphyrins (Dolphin, D., ed.), Vol. VII, pp. 333-375, Academic Press, New York.

2. WHITE, R. E., AND COON, M. J. (1980) Anna Rev. Biochem. 49,315-356.

3. RAHIMTULA, A. D., AND O’BRIEN, P. J. (1975) Biochem. Biophys. Res. Commun. 62,268-275.

4. NORDBLOM, G. D., WHITE, R. E., AND COON, M. J. (1976) Arch. Biochem. Biophys. 175,524- 533.

5. CAPDEVILA, J., ESTABROOK, R. W., AND PROUGH, R. A. (1980) Arch. Biochem. Biophys. 200,186- 195.

6. GRIFFIN, B. W., MARTIN, C., YASUKOCHI, Y., AND MASTERS, B. S. S. (1980) Arch. B&hem. Bio- phys. 205,543-553.

7. GRIFFIN, B. W., AND TING, P. L. (1978) Biochem- istry 17, 2266-2211.

8. HEWSON, W. D., AND HAGER, L. P. (1979) in The Porphyrins (Dolphin, D., ed.), Vol. VII, pp. 295- 332, Academic Press, New York.

9. HOLLENBERG, P. F., AND HAGER, L. P. (1973) J. BioL Ch.em. 248.2630-2633.

10. GUNSALUS, I. C., MEEKS, J. R., LIPSCOMB, J. D., DEBRUNNER, P., AND M~~NcK, E. (1974) in Mo- lecular Mechanisms of Oxygen Activation (Hayaishi, 0.. ed.), pp. 559-613, Academic Press, New York.

11. ORRENIUS, S., AND ERNSTER, L. (1974) in Molec- ular Mechanisms of Oxygen Activation (Hay- aishi, O., ed.), pp. 215-244, Academic Press, New York.

12. KEDDERIS, G. L., KOOP, D. R., AND HOLLENBERG, P. F. (1980) .I BioL Chem. 255,10,174-10,182.


13. GRIFFIN, B. W. (1978) Arch. B&hem. Biophys. 190,850~853.

14. KAHN, G. C., BOOBIS, A. R., BLAIR, I., BRODIE, M. J., AND DAVIES D. S. (1980) Brit. J. Clin Pharmacol 9, 284P.

15. KELLERMANN, G. H., AND LUYTEN-KELLERMANN, M. (1978) Life Sci. 23,2485-2490.

16. MORRIS, D. R., AND HAGER, L. P. (1966) J. Biol. Chem. 241.1’763-1768.

17. HAGER, L. P., MORRIS, D. R., BROWN, F. S., AND EBERWEIN, H. (1966) J. BioL Chem. 241,1769- 1777.

18. HOLLENBERG, P. F., RAND-MEIR, T., AND HAGER, L. P. (1974) J. BioL Chem. 249, 5816-5825.

19. NELSON, D. P., AND KIESOW, L. A. (1972) AnaL Biochem. 49,474-478.

20. NASH, T. (1953) B&hem. J. 55.416-421. 21. RAMEITE, R. W., AND SANFORD, R. W. (1965) J.

Amer. Chem. Sot. 87,5001-5005. 22. THOMAS, J. A., MORRIS, D. R., AND HAGER, L. P.

(1970) J. BioL Chem. 245,3135-3142. 23. PERKOV, W. (1963) U. S. Pat. No. 3,092,483, Chem.

Abstr. 59.13999b. 24. GALVEZ, C., ALBALA, J., AND MESA, J. (1973) An.

Quim. 69,1319-1324. 25. FUSON, R. C. (1962) Reactions of Organic Com-

pounds, p. 170, Wiley, New York. 26. TOM~NKOV~, H., AND Z?KA, J. (1975) Micro&em.

J. 20, 132-153. 27. AWE, W., STOY-GEILICH, E., BAUERHOP, R., AND

BAMMEL, H. (1960) Arzneim Forsch. 10, 796- 800.

28. PECHTOLD, F. (1964) Arzneim. For.& 14, 258- 259.

29. HAGER, L. P., DOUBEK, D. L., SILVERSTEIN, R. M., LEE, T. T., THOMAS, J. A., HARGIS, J. H., AND MARTIN, J. C. (1973) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S., and Morrison, M., eds.), Vol. I, pp. 311-327, Univ. Park Press, Baltimore, Md.

30. MORRISON, M., AND SCHONBAUM, G. R. (1976) Annu. Rev. B&hem. 45.861-888.

31. THOMAS, J. A. (1968) Ph. D. Dissertation Univ. of Illinois, Urbana, Ill.

32. DOWNS, A. J., AND ADAMS, C. J. (1973) in Com- prehensive Inorganic Chemistry (Bailar, J. C., Jr., Emeleus, H. J., Nyholm, R., and Trotman- Dickerson, A. F., eds.), Vol. 2, pp. 1480-1490, Pergamon Press, Oxford, England.

33. LEWIN, M. (1966) in Bromine and its Compounds (Jolles, Z. E., ed.), pp. 703-729, Academic Press, New York.

34. SILVERSTEIN, R. M., AND HAGER, L. P. (1974) Bio- chemistry 13.5069-5073,

35. VESELL, E. S. (1977) in Microsomes and Drug Oxidations (Ullrich, V., Hildebrandt, A., Roots, I., Estabrook, R. W., and Conney, A. H., eds.) pp. 628-645, Pergamon Press, Oxford.

36. DANHOF, M., KROM, D. P., AND BREIMER, D. D. (1979) Xenobiotica 9,695-702.

37. CORBETT, M. D., CHIPKO, B. R., AND BATCHELOR, A. 0. (1980) Biochem. J. 187,893-903.

38. Lu, A. Y. H., JACOBSON, M., LEVIN, W., WEST, S. B., AND KIJNTZMAN, R. (1972) Arch. Biochem. Biophys. 153.294297.

39. SMITH, M. R., AND GORROD, J. W. (1978) in Bio- logical Oxidation of Nitrogen (Gorrod, J. W., ed.), pp. 65-70, Elsevier/North Holland, Am- sterdam.

40. ADAMS, R. N. (1969)Accts. Chem, Res. 2,175-180. 41. ASHLEY, P. L., DAVIS, D. K., AND GRIFFIN, B. W.

(1980) B&hem. Biophys. Res. Commun. 97, 660-666.

42. HARRISON, J. E., AND SCHULTZ, J. (1976) J. BioL C&m. 251,1371-1374.

43. ROSENBLATT, D. H., HALL, L. A., DELUCA, D. C., DAVIS, G. T., WEGLEIN, R. C., AND WILLIAMS, K. K. R. (1967) J. Amer. Chem. Sot. 89,1158- 1162.

44. DE LA MARE, H. E. (1960) J. Org. Chem. 25,2114- 2126.

45. RENARD, J. J., AND BOLKER, H. I. (1976) Chem. Rev. 76,487-508.

46. INGOLD, K. U. (1973) in Free Radicals (Kochi, J. K., ed.), Vol. 1, pp. 37-112, Wiley, New York.

47. BERLINER, E. (1966) J. Chem. Ed 43,124-133. 48. POUTSMA, M. L. (1973) in Free Radicals (Kochi,

J. K., ed.), Vol. II pp. 159-229, Wiley, New York.

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Chloroperoxidase-catalyzed halogenation of antipyrine, a drug substrate of liver microsomal cytochrome P-450