nitroacetophenone - The Journal of Biological Chemistry

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Feb 6, 1986 - Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, ... Immunology, Beckman Research Institute of the City of Hope, ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 261, No. 25, Issue of September 5 , pp. 11478-11486,1986 Printed in U.S. A.

Chemical Modificationand Inactivationof Rat Liver Microsomal Cytochrome P-45Oc by 2-Bromo-4’-nitroacetophenone* (Received for publication, February 6, 1986)

Andrew Parkinson$, Dene E. Ryan, PaulE. Thomas, DonaldM. Jerinas, JaneM. Sayer$, Peter J. van Bladeren$, MitsuruHaniull, John E. Shivelyn, and Wayne Levin11 From the Laboratory of Experimental Carcinogenesis and Metabolism, Roche Institute of Molecular Biology, Nutley, New Jersey 071IO, the §Laboratory of Bioorganic Chemistry, Section on Oxidation Mechanisms, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland20892, and the (Divisionof Immunology, Beckman Research Instituteof the City of Hope, Duarte, California 91010

The alkylating agent 2-bromo-4’-nitroacetophenone of rat liver microsomal cytochromeP-450 (designated P-450a(BrNAP) binds covalently to each of 10 isozymes of P-45Oj) have beenpurified in our laboratory and characterized purified rat liver microsomal cytochrome P-450 (P- with respect to their role in xenobiotic biotransformation and 450a-P-450j) but substantially inhibits the catalytic their regulation by various physiologic and environmental activity of only cytochromeP-45Oc. Regardless of pH, factors (%lo).’ incubation time, presenceof detergents, or concentraThe ability of cytochrome P-45Oc to catalyze the stereosetion of BrNAP, treatment of cytochrome P-45Oc with lective metabolism of a wide variety of polycyclic aromatic BrNAP resulted in no more than 90% inhibition of hydrocarbons has been used to determine the spatial tolerance catalyticactivity.AlkylationwithBrNAPdid not of the substrate-binding site of this 3-methylcholanthrenecause the release of heme from theholoenzyme or alter inducible hemeprotein (22,23).Although theamino acid the spectral properties of cytochrome P-450c,data that sequence of cytochrome P-45Oc has been determined (16,17), exclude the putative heme-binding cysteine, Cys-460, those amino acids that comprise the catalytic site and define as the major site of alkylation. Two residues in cytothe boundary of the substrate-binding site have not been chrome P-45Oc reacted rapidly with BrNAP, for which reason maximal lossof catalytic activity was invaria- established. We have now undertaken chemical modification bly associated with the incorporation of -1.5 mol of studies toidentify functionally important aminoacid residues BrNAP/mol of cytochrome P-45Oc. Two major radio- in cytochrome P-450 as an approach to gain further insight labeled peptides were isolated from a tryptic digestof intothemechanism of catalysis by thisunusual class of enzymes. [14C]BrNAP-treated cytochrome P-45Oc by reverseInasmuch as cytochrome P-45Oc is noted for its efficient phase high performance liquid chromatography. The amino acid sequence of each peptide was determined metabolism of polycyclic aromatic hydrocarbons (22-24), we by microsequence analysis,but theidentification of the had initially considered synthesizing a bromoacetyl derivative residues alkylated by BrNAP was complicated by the of such substrates in an attempt to alkylate one or more tendency of the adducts todecompose when subjected amino acid residues in the catalytic/substrate-bindingsite. to automated Edman degradation. However, results of However, since we had previously identified a histidine resicompetitive binding experiments with the sulfhydryl due in the catalytic site of epoxide hydrolase with BrNAP’ reagent4,4’-dithiodipyridineidentifiedCys-292 as (25), we initiated the present studies on the interaction bethe major site of alkylation and Cys-160as the minor tween the alkylating agent, BrNAP, and each of 10 isozymes site of alkylation by BrNAP in cytochromeP-45Oc. of highly purified rat liver microsomal cytochrome P-450, as well as NADPH-cytochrome P-450 reductase. EXPERIMENTALPROCEDURES

Cytochrome P-450 refers to a family of membrane-bound hemeproteins that function as monooxygenases in the biotransformation of numerous xenobiotics (e.g. drugs, carcinogens,pesticides, etc.) andendogenoussubstrates,suchas steroids, fatty acids, and certain vitamins (1).Ten isozymes * This work was supported in part by the Speas Foundation and by National Institutes of Health Grants ES 03765 (A. P.) and GM 34426 (M. H.). A preliminary account of this work was presented a t the Sixth International Symposium on Microsomes and Drug Oxidations, Brighton, England, August, 1984 and in abstract form (Parkinson, A., Ryan, D. E., Thomas, P. E., Jerina, D.M., Haniu, M., Shively, J. E., and Levin, W. (1985) Fed. Proc. 44, Abstr. 7024). The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Dept. of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center,Kansas City, KS 66103. 11 To whom reprint requests should be addressed.

Chemicak-BrNAP was obtained from Eastman and recrystallized from ethanol before use. 4-Nitroacetophenone, bromoacetic acid, 2bromoacetophenone, and PDS were purchased from Aldrich, 2-bromoacetamido-4-nitrophenolfrom Sigma, and l-chloro-2,4-dinitrobenzene and 2-bromo-2-phenylacetophenone from Eastman. Sodium cholate, CHAPS, octyl glucoside, dilauroylphosphatidylcholine, and The term cytochrome P-450 is used to refer to any or all forms of liver microsomal cytochrome P-450. Because a nomenclature for the various forms of cytochrome P-450 has not been established, we designate the rathemeproteins based on the order in which they were purified. Rat liver cytochromes P-450a-P-450j have different amino acid sequences (7, 11-21). Cytochrome P-45Oc is inducible in liver microsomes by as much as 50-fold when rats are administered 3methylcholanthrene (8,9) or certain other xenobiotics (9, 10). The abbreviations are: BrNAP, 2-bromo-4’-nitroacetophenone; [I4C]BrNAP,2-bromo-4’-nitro[carbonyl-’4C]acetophenone;TPCK, L1-p-tosylamido-2-phenylethyl choloromethyl ketone; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate;DTT, dithiothreitol; HPLC, high performance liquid chromatography; octyl PDS, 4,4’-dithiodipyridine. glucoside, octyl-j3-D-glucopyranoside;

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Chemical Modification of DTT were obtained from Calbiochem, and Emulgen 911 was from Kao-Atlas Co., Tokyo, Japan. All organic solvents were purchased from Burdick and Jackson. Synthesis of Bromoacetyl Compounds-["C]BrNAP (5.6 Ci/mol) was synthesized from 4-nitro[~arboxyl-'~C]ben~oic acid as previously described (25).Thin layer chromatography of the compound indicated a radiochemical purity of >97%. 2-(2-Bromoacetyl)-naphthalene, 9(2-bromoacetyl)-anthracene, 2-(2-bromoacetyl)-fluoreneand 2-, 3-, were obtained by bromination and 9-(2-bromoacetyl)-phenanthrene of the corresponding acetyl hydrocarbons according to the general procedure of May and Mosettig (26). Typically, 1g of acetyl derivative in 30 ml of ether was cooled in an ice bath, and 1eq of bromine in 5 ml of dichloromethane was added with stirring over a 10-min period. After additional stirring for 50 min, solvents were removed under vacuum, and the residues were dissolved in benzene. The benzene solutions were passed through a small bed of Florisil and thedesired products crystallized from benzene/hexane. Structures and purities were established by NMR and mass spectroscopy. Purification of Microsomal Enzymes-Cytochromes P-450a (2), P450b, P-450c, and P-450e (4) were purified as described from liver microsomes of Aroclor 1254-induced Long-Evans rats; cytochrome P-450d from isosafrole-induced rats (3); cytochromes P-450f, P-450g, P-450h, and P-450i from untreated rats (5, 6), and cytochrome P450j from isoniazid-treated rats (7). Each form of cytochrome P-450 was electrophoretically homogeneous in sodium dodecyl sulfate-polyacrylamide gels with a specific content of a t least 12 nmol of cytochrome P-450/mg of protein. NADPH-cytochrome P-450 reductase was isolated by a combination of the methods of Dignam and Strobe1 (27) and Yasukochi and Masters (28). The purified flavoprotein had a specific activity of 39,000 units/mg of protein determined with cytochrome c a t 22 "C as described by Phillips and Langdon (29). Dilauroylphosphatidylcholine was used as anaqueous solution which wassonicated immediately before use. Monoclonal Antibody Production-Production, purification, and characterization of monoclonal antibody C8 against cytochrome P450c have been described by Thomas et al. (30). This monoclonal antibody recognizes a single polypeptide ( k t r = 56,000) on "Western blots" of rat liver microsomes, selectively and completely inhibits the catalytic activity of cytochrome P-450c, and does not react with the other nine cytochrome P-450 isozymes used in this study (30). Regeneration of Free Sulfhydryl Groups-We have shown previously that thefree sulfhydryl groups of cytochrome P-450 can oxidize to form disulfides during long-term storage at -80 "C (31). To eliminate this possibility, each cytochrome P-450 isozyme (typically 5 nmol/ml of 50 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 100 PM EDTA) was treated under nitrogen with a 10-foldmolar excess of sodium sulfite for 15 min at room temperature, followed by a 100-fold molar excess of DTT for 15 min at room temperature (31). To remove the excess sulfite and DTT, theprotein was dialyzed overnight at 4 "C against 2 X 2 liters of potassium phosphate buffer (100 mM, pH 7.4) containing 20% glycerol. This procedure had no effect on the catalytic or spectral properties of the proteins. An aliquot of the dialyzed sample was diluted 10-fold in the same buffer containing 0.5% sodium cholate and 0.2% Emulgen 911, and theconcentration of cytochrome P-450 determinedby the method of Omura and Sat0 (32). The remainder of the dialyzed sample was diluted with potassium phosphate buffer (100 mM, pH 7.4) to 1 nmol of cytochrome P-450/ml, unless stated otherwise. Chemical Modification with BrNAP-Unless otherwise indicated, cytochrome P-450 or NADPH-cytochrome P-450 reductase (1p ~ in) potassium phosphate buffer (100 mM, pH 7.4)was treated with BrNAP (5 or 50 p ~ or) solvent (acetonitrile, 2.5%) for 30 min at room temperature, at the final concentrations indicated. Unless otherwise stated, reactions were terminated with DTT (100-200 p ~ ) and aliquots of the samples were assayed for catalytic activity as described below. HPLC analysis established that DTTreacts rapidly with BrNAP to form an adduct. When ["CIBrNAP was used, the degree of covalent binding was determined as previously described (25) with minor modifications. Briefly, aliquots (100 pl) of["C] BrNAP-treated enzyme were applied to 24-mm Whatman glass filter discs (GF/A), air dried, and washed for 10 min with ice-cold 20% trichloroacetic acid (2 X 100 ml), methanol (2 X 100 ml), and anhydrous diethyl ether (50 ml). The filters were dried and transferred to scintillation vials containing 12 ml of Aquasol (New England Nuclear). Radioactivity was measured in a Packard Tri-Carb spectrometer with 86% countingefficiency. Under these conditions, the binding of 0.1 nmol of ['4C]BrNAP/0.1 nmol of enzyme gave values of

Rat Cytochrome P-45Oc

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-1100 cpm, compared to 97% of the radioactivity from the aqueous phase a t each time point) was applied to theprebinder region of Whatman LK5DF silica TLC plates. To facilitate the localization of unchanged [14C]BrNAP under uv light, unlabeled BrNAP was applied to the TLC plates, which were then developed in benzene. The region of the plate containing BrNAP was scraped directly into a scintillation vial, and radioactivity was determined. To determine the apparent solubility of BrNAP in aqueous solution, 10 pl of 85 mM [I4C]BrNAP(dissolved in acetonitrile)was added to 0.4 ml of potassium phosphate buffer (100 mM, pH 7.4) at room temperature. Insoluble ["CIBrNAP was removed by centrifugation followedby filtration through Whatman No. 1 filter paper. From measurements of the radioactivity in the clear filtrate, the apparent solubility of BrNAP was estimated to be -600 p ~ . Release of 4-Thiopyridone from PDS-treated Cytochrome P-45OcCytochrome P-45Oc that had been treated with sodium sulfite, DTT, and exhaustively dialyzed was treated with 2 molof PDS/mol of cytochrome for 30 min at room temperature. The sample was then dialyzed overnight against 2X 2 litersof 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol.PDS-treated cytochrome P450c (5 nmol), with or without reconstitution with 16 nmol of NADPH-cytochrome P-450 reductase and 240nmol of dilauroylphosphatidylcholine, was incubated a t 4 "C for 5 min in atotal volume of 5 ml containing 1mM NADPH, 100 mM potassium phosphate (pH 7.4) and 3 mM MgC12. Following the incubation, 1.64 mlof acetone and 0.5 mlof l-chloro2,4-dinitrobenzene (0.1 M in acetone) were added to each 5-ml incubation mixture, and theresultant solutions were incubated for 1 h at 37 "C. The mixtures were cooled and acidified to pH -2 with 1 ml of 1 M hydrochloric acid and washed 3 times with 2-ml portions of hexane to remove most of the unreacted l-chloro-2,4-dinitrobenzene. The combined hexane solutions were extracted with 1-ml portionsof 0.01 M hydrochloric acid, whichwere combined with the aqueous phase from the hexane extraction. This acidic aqueous solution, which contained the trapped product, 1-(4-pyridinethi0)-2,4-dinitrobezene, in the protonated form was adjusted to pH -9-10 with 1.15 ml of 1 M sodium hydroxide and extracted 3 times with 2-ml portions of ethyl acetate. The ethyl acetatesolution was dried with sodium sulfate and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 0.2 ml of methanol and 0.2 ml of water, and 50- or 100pl samples were analyzed by HPLC on a Perkin-Elmer C18 column (3 p ) eluted with methanol, 0.05 M Tris-acetate buffer (pH 7), 45:55, at a flow rate of 2 ml/min. Under these conditions the trappedproduct is eluted a t from 4-thiopyridone, l-(pyridinethio)-2,4-dintrobenzene), 3.3 min. 4-Thiopyridone was quantified by integration of this peak area, detected a t 330 nm, and comparison with astandard line generated by injection of dilutions (containing 0.5-2.0 nmol of product) of a mixture of authentic 4-thiopyridone (92 p ~ and ) 7 mM 1chloro-2,4-dinitrobenzenethat had been allowed to react under the conditions described above. Reproducibility of injections was ensured by the use of aPerkin-Elmer ISS-100 autosampler. In separate experiments, the trapped product was shown to be extracted, quantitatively by the procedure described, and Emulgen 911, dilauroylphosphatidylcholine and NADPH, at the concentrations used in the enzyme studies, did not interfere with its detection or quantitation. The identity of the product derived from the PDS-treated enzyme , with that derived from authentic 4-thiopyridone was verified by measurement of on-line UV spectra of the chromatographic peaks (with an LKB model 2140diode-array detector) and by measurement of the mass spectra of the eluted compounds after extraction into ethyl acetate (CI,NH3); m/z of base peak 278 (M 1). Catalytic Actiuity-The hydroxylations of aniline, benzo[a]pyrene, [4,6-3H]zoxazolamine, [4-"C]testosterone, and [1,2-3H]5P-androstane-3~~,17&disulfate, the 0-dealkylation of 7-ethoxycoumarin, and the N-demethylation of [N-methyl-'4C]benzphetamine.HCl were determined as described (Ref. 7 and references therein). The catalytic activity of each cytochrome P-450 isozyme was measured at 37 "C in a reconstituted system containing saturating amounts of NADPHcytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine, as described (2-7). Each cytochrome P-450 isozyme

+

11480

Chemical Modification Cytochrome of Rat

was assayed forcatalytic activity under optimal conditions such that productformationwas directly proportional to cytochromeP-450 concentration and incubation time. Initial experiments established that neither BrNAP northe BrNAP-DTT adduct interfered withthe detection or stability of the reaction products. The catalytic activityof NADPH-cytochrome P-450 reductase was measured by two methods.In the first, activity was measuredat 22 'C by the rate of reduction of cytochrome c, as described byPhillips and Langdon (29). In the second method, activitywas measured at 37 "C by the ability of the flavoprotein to support the cytochrome P-45Ocdependent hydroxylationof benzo[a]pyrene ina reconstituted system containing 0.014, 0.04, or 0.22 nmol of NADPH-cytochrome P-450 reductase, 0.02nmol of cytochrome P-450c, and 31 nmol of dilauroylphosphatidylcholine in 1 ml of potassium phosphate buffer (100 mM, pH 7.4) containing 3 mM MgCl, and 500 p M NADPH. MicrosequenceAnalysis-To identify the aminoacidresidue(s) alkylated by BrNAP, cytochrome P-45Oc (5 p ~ was ) treated with sodium sulfite and DTT toregeneratefreesulfhydrylgroups(see above) and dialyzed overnight at 4 "C against potassium phosphate buffer (100 mM, pH 7.4) containing 20% glyceroland 100 F M EDTA. The dialyzed sample was diluted to 2 nmol of cytochrome P-450c/ml with the same buffer and dividedinto 2 X 6-ml aliquots,one of which was treated with 24 nmol of the sulfhydryl reagent, PDS, at room temperature for 30 min. The untreated and PDS-treated cytochrome P-45Oc samples were dialyzedat 4 "C against 2 X 1liter of potassium phosphate buffer (100mM, pH 7.4) containing 20% glycerol and 100 p~ EDTA and diluted with the same buffer to 1 p~ cytochrome P450c. Both samples weretreated with [WIBrNAP (5 p M ) for 30 min at room temperature, and the reactions were terminated with 25 p~ DTT. Initial experiments establishedthat glycerol, which was usually absent from the incubation mixtures, hadno effect on the alkylation of cytochromeP-45OcbyBrNAP.After aliquots wereremoved to measure catalytic activity and covalent binding of BrNAP to cytochrome P-450c, both samples were dialyzed overnightat 4 "C against 2 X 1liter of ammonium bicarbonate (200mM, pH 8.0). After dialysis, the concentration of cytochrome P-45Oc (which was not denatured by this treatment) was approximately 0.75 pM. After an aliquot of the dialyzed samples was removedto measure covalent binding, both the BrNAP-treatedcytochrome P-45Ocand the BrNAP-treated PDSpretreated cytochrome P-45Oc were digested for 24 h at 37 "C with TPCK-treated trypsin (enzyme-to-protein ratio,1:50, w/w) (18).The tryptic peptides (from-10 nmol of cytochrome P-45Oc) were resolved by reverse-phaseHPLCasdescribed(18)onanUltrasphere C-8 column (250 X 4.6mm,5-pm particle size) with a linear gradient from 100% solvent I (0.1% trifluoroacetic acetic acid)to 60% solvent I1 (trifluoroacetic acidwater:acetonitrile,1:99:900) over 3 h at a flow rate of 0.8 ml/min. Peptides were detected by absorbance at 206 nm and collected manually. The radioactivity in an aliquot (20 pl) of each fraction was determined to identify those peptides containing amino acid residues alkylated with [14C]BrNAP. The amino acid sequence of peptides alkylated with ["CIBrNAP was determined by automated Edman degradation witha gas phase sequenator as described (33). The phenylthiohydantoins were identified by reverse-phase HPLC on an Altex Ultrasphere ODS column (34). RESULTS ANDDISCUSSION

Alkylation of Cytochrome P-450 Isozymes by BrNAP-The effects of treating each of 10 isozymes of highly purified cytochrome P-450 or NADPH-cytochrome P-450 reductase with a 5- or 50-fold molar excess of [14C]BrNAP are shown inTable I. TreatmentwithBrNAP for30 min at room a concentration-dependent alkylation temperature resulted in of each enzyme tested. The degree of alkylation (mol of BrNAP bound permol of enzyme) with 5 PM BrNAP ranged from 0.6 (cytochrome P-45Ob) to 2.1 (cytochrome P-45Og) 1.3 whereaswith 50 PM BrNAP,alkylationrangedfrom (cytochrome P-450a) to3.7 (cytochrome P-45Og). Although BrNAP alkylated eachisozyme of cytochrome P450 tested to a comparable degree, only cytochrome P-45Oc suffered a substantial loss of catalytic activity (>80%) when treated with a &fold molar excess of BrNAP. Cytochrome P450g exhibited a 46% loss of catalytic activity butonly when treated with a 50-fold molar excess of BrNAP (under which

P-45Oc

conditions 3.7 mol of BrNAP were incorporated per mol of cytochrome P-45Og). The catalytic activity of the other 8 isozymes of cytochrome P-450 treated with 5 or 50PM BrNAP was unaffected (ie. altered by less than 10%) or was stimulated(upto 63% asinthe case of cytochrome P-450h). Interestingly, cytochromeP-450d, which isimmunochemically (30, 35) related to and shows 68% total sequence homology (16, 17, 19) with cytochrome P-450c, was among the hemeproteins whose catalytic activity was stimulated by alkylation with BrNAP. It is important to note that the catalytic activity of the 10 isozymes of cytochrome P-450 listed in Table I cannot be evaluated adequately with a single substrate (or a substrate that is converted by each enzyme to a common product) (2selective inactivation 7). It could be argued, therefore, that the of cytochrome P-45Oc by BrNAP is dependent on the substrate rather than the hemeprotein under investigation. To test this possibility, benzo[a]pyrene was also used as a substrate to measure the catalytic activity of cytochrome P-450b before and after treatment with a 50-fold molar excess of BrNAP. Table I shows that alkylation with BrNAP slightly activated the catalytic activityof cytochrome P-450b toward benzphetamine andbenzo[a]pyrene. Additionally, zoxazolamine was used as a substrate for both cytochromes P-45Oc and P-450d, and the results (Table I) confirmed that theselective inactivation of cytochrome P-45Oc is unrelated to the substrate but reflects instead a property unique to cytochrome P-450~. Treatment with BrNAP for 30 min at room temperature also resultedin a concentration-dependentalkylation of NADPH-cytochrome P-450 reductase, the extent of which was comparable to the cytochrome P-450 isozymes tested (Table I). Treatment of NADPH-cytochrome P-450 reductase with 50 PM BrNAP (but not 5 PM) caused a substantial loss of catalytic activity, determined either by the rateof reduction of cytochrome c or by the hydroxylation of benzo[a]pyrene in a reconstituted system containing cytochrome P-45Oc and dilauroylphosphatidylcholine.The reason why alkylation of NADPH-cytochrome P-450 reductase inhibited catalytic acc (83% inhibition) more than toward tivity toward cytochrome cytochrome P-450c/benzo[a]pyrene (58% inhibition) is unknown, although it has long been known that the ability of NADPH-cytochrome P-450 reductase reduce to artificial electron acceptors, such as cytochrome c, can bedissociated from its ability toreduce cytochrome P-450 (36). It should be emphasized thatresidual BrNAP in alkylated preparations of cytochrome P-45Oc cannot inactivate NADPH-cytochrome P-450 reductasewhen the two proteins arereconstituted. Cytochrome P-45Oc was treatedwith BrNAP and combined with the flavoprotein only after the excess BrNAP was alkylated with DTT or removed by exhaustive dialysis. Even if some of the excess BrNAP survived these treatments, the 10-50-fold dilution of cytochrome P450c in the reconstituted system would lower the BrNAPlevel well below that required to inactivate NADPH-cytochrome P-450 reductase (see Table I). Furthermore, all cytochrome P-450 isozymes would sufferaloss of catalytic activity if NADPH-cytochrome P-450 reductase became alkylated and inactivated by any residual BrNAP during the reconstitution experiments. Spectral Properties of Alkylated Cytochrome P-450c-The effects of alkylation with BrNAP on the spectral properties of cytochrome P-45Oc were determined to examine the possibility that BrNAP inactivatedcytochrome P-45Oc simply by disrupting theheme moiety of cytochrome P-45Oc. Both nonalkylated and alkylated cytochrome P-45Oc displayed an ab-

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Chemical Modification ofRat Cytochrome P-45Oc TABLEI Effect of treatment with BrNAP on the catalytic activity of purified cytochrome P-450 isozymes and NADPHcytochrome P-450 reductase Cvtochrome P-450 isozvmes and NADPH-cvtochrome P-450 reductase (1 UM) were treated with 5 or 50 U M ["CjBrNAP for 30 min a t 22"C, and the reactions were terminated by the addition of 200 p~ DTT. Aliquots' of the samples were assayed for catalytic activity and covalent binding as described under "Experimental Procedures." Catalytic activity ;Y ,~',,an,,,',g remaining 51" 501"

Covalent binding Protein

Substrate

Keaction Reaction 5:1"

501"

mollmol protein

%

103 1.3 0.9 7a-Hydroxylation Testosterone 114 1.6 0.6 N-Demethylation Benzphetamine 3- and 9-Hydroxylation Benzo[a]pyrene 2.9 1.6 18 3- and 9-Hydroxylation Benzo[a]pyrene P-450~ 10 6-Hydroxylation Zoxazolamine 9 0-Deethylation 7-Ethoxycoumarin 131 1.1 2.2 6-Hydroxylation Zoxazolamine P-450d 2.3 0.9 111 N-Demethylation Benzphetamine P-450e 129 2.0 16a-Hydroxylation 3.6 Testosterone P-450f 2.1 3.7 117 Testosterone 6p- and 15a-Hydroxylation P-45Og 3.5 1.9 163 N-demethylation Benzphetamine P-450h 1.5 107 15fi-Hydroxylation 3.0 Androstane disulfate P-450i 2.3 1.0 103 4-Hydroxylation Aniline P-450j 1.1 3.2 86 Reduction Cytochrome c Reductase 90' 3- and 9-Hydroxylation Cytochrome P-450c/ benzolalpyrene Molar ratio of BrNAP. * In theabsence of BrNAP, the turnover numbers (nmol of product formed/nmol of cytochrome P-450/min) of each isozyme tested were comparable to previously published values (2-7). e Values are the mean of determinations a t three different molar ratios of reductase to cytochrome P-45Oc (11, 2, and 0.7) in the reconstituted system. In all cases, the percent inhibition of catalytic activity by BrNAP was virtually identical. P-450a P-450b

sorbance maximum at 447 nm in the carbon monoxide difference spectrum of the dithionite-reduced hemeprotein, indicating that alkylation with BrNAP did not convert cytochrome P-45Oc to cytochrome P-420. Like the wavelength "), of the carbon monmaximum, the peak height (~M~~7-490 oxide difference spectrum of ferrous cytochrome P-45Oc was unaffected by alkylation of the hemeprotein with BrNAP, indicating that treatment with BrNAP did not cause the loss of heme from cytochrome P-45Oc. Ferrous nonalkylated and alkylated cytochrome P-45Oc also produced identical difference spectra with ethyl isocyanide, with an absorbance maximum at 452 nm and apeak height ratio (452/430 nm) of >1.7 at pH 7.4. In the absolute spectrum, both nonalkylated and alkylated cytochrome P-45Oc absorbed light maximally at 417 nm with an extinction coefficient of 127 mM" cm", indicative of a low spin-ferric state for both hemeproteins. Inasmuch as the ligand-binding characteristics of cytochrome P-450 are dependenton an intact heme-thiolate bond (37, 38), the unaltered ligand-binding properties make it extremely unlikely that BrNAP alkylates Cys-460, the cysteine residue that putatively serves as the fifth ligand of the heme iron (17, 39, 40). Since the sixth ligand to the heme iron is thought to be important in determining the spin state of cytochrome P-450 (37, 38), the unaltered spin state of alkylated cytochrome P-45Oc suggests that BrNAP does not alkylate the (as yet unidentified) sixth ligand of the heme iron. The results of these spectral studies indicate that the inactivation of cytochrome P-45Oc by BrNAP does not simply involve a conversion to cytochrome P-420 or a dissociation of the heme moiety from cytochrome P-450c, but apparently involves the alkylation of a residue(s)that alters thecatalytic effectiveness of cytochrome P-45Oc. The Effects of BrNAP Concentration and Time of Incubation-The relationship between the loss of catalytic activity toward benzo[a]pyrene and the covalent binding of BrNAP to cytochrome P-450c, as a function of the concentration of

I

1

I

l,kl

I

I

1

99 129 137 13 9 9 128 117 110 54 139 102 91 17 42'

I

0 2 4 6 8 IO 20 40 60 80 100 MOLAR RATIO OF &NAP TO CYTOCHROME P450c

FIG. 1. Inactivation and alkylation of cytochrome P-45Oc as a function of BrNAP concentration. Cytochrome P-45Oc (1 p ~ was ) treated with up to a 100-fold molar excess of ["CIBrNAP for 30 min at 22 "C, and thereactions were terminated by the addition of 200 p~ DTT. Aliquots of the samples were assayed for catalytic activity and covalent binding as described under "Experimental Procedures."

BrNAP, is shown in Fig. 1. Treatment of cytochrome P-45Oc (1 FM) with various concentrations of BrNAP for 30 min at room temperature resulted in alinear loss of catalytic activity up to 3 PM BrNAP. Cytochrome P-45Oc treated with 5 FM BrNAP lost -85% of its catalyticactivity, and no further loss was observed when cytochrome P-45Oc was treated with up to 100 F M BrNAP. Although catalytic activity was maximally inhibited by treatment with 5 PM BrNAP, the covalent binding of BrNAP tocytochrome P-45Oc continued to increase up to at least 100 PM BrNAP. Covalent binding was directly proportional to the concentration of BrNAP up to 3 p~ (i.e. a doubling of concentration up to 3 p~ BrNAP increased the covalent binding 2-fold).

Chemical Modificationof Rat Cytochrome P-45Oc

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TABLEI1 The effect of various chemicals on the catalytic activityof cytochrome P-45Oc Cytochrome P-45Oc (1 p M ) was pretreated with various chemical reagents or the solvent (acetonitrile, 2.5%final concentration) for 30 min at 22 “C. An aliquot was removed, treated with 200 ~ L MDTT, and the sample was analyzed for benzo[a]pyrene metabolism. The remainder of the sample was treated with [“CIBrNAP (5 mol/mol of cytochrome P-45Oc) for 30 min at 2 2 T , and the reaction was terminated with 200 p~ DTT. Covalent binding was determined as described under “Experimental Procedures.” PREIREATMENT molar ratio

compound

ACT” BOUND [‘%ErNW perce?t moVmol remoinlng cytochrome P-45Oc 100

Solvent

1.5-1.7

PRETREATMENT molar compound mtia Solvent

100

1.5-1.7

94

1.6

43 47

0.3 0.4

36

42

0.2 0.2

34

0.3

7.e

y2%

c =o

7-0

101

15-21

0.3

101 0.N

No2

$w

2-Eromoa&mido4-nitmDh.nal

B r W

k

ACT” BOUND r‘4ClErN4F’ percent moVmol remaining cytochrome P-45Oc

2

4-Nitroocetophenone 0

101 5 01

91 98

1.6 1.6

2-(2-Eromwcdyl)-fluonn.

Bromoocetic Acid CHZ&

101 5 01

39 22

0.5 0.2

2-Bromoacetophenone

3-(2-Emmw~l)-phenanthr~e 0

&

22 14

0.3 0.1

68 77

1.3 1.5

0

51 00 1

The results of experiments on the alkylation of cytochrome P-45Ocby BrNAP were not significantly influenced by the half-life or the solubility of the alkylating agent in aqueous solution. Measurement of the stability of [14C]BrNAP in potassium phosphate buffer (100 mM, pH 7.4) indicated the alkylating agent decomposed with a half-life of 4.8 h; hence, less than 10% of the BrNAP would have decomposed during the course of a 30-min incubation. The apparentsolubility of [14C]BrNAPin potassium phosphate buffer (100 mM,pH 7.4) at 22 “C was determined to be -600 p ~ as, described under “Experimental Procedures.” This value far exceeds the highest concentration of BrNAP tested (i.e. 100 p ~ ) . Three important findings are illustrated in Fig. 1.First, the loss of catalytic activity toward benzo[a]pyrene and the covalent binding of BrNAP to cytochrome P - 4 5 0 ~show good correspondence only at very low concentrations of BrNAP ( ( 5 p ~ ) Second, . treatment of cytochrome P-45Oc for 30 min at room temperature does not lead to a complete loss of catalytic activity. Even cytochrome P-45Oc treated with 100 p~ BrNAP (i.e. 20 X the concentration effecting maximal inactivation) retained -15% of its catalytic activity toward benzo[a]pyrene. Third, maximal loss of catalytic activity was associated with the incorporation of approximately 1.5 mol of BrNAP/mol of cytochrome P-45Oc. This latter finding indicates that at least two amino acid residues in cytochrome P450 are extremely reactive toward BrNAP, at least one of

101 -1

58

0.2

which is responsible for the loss of catalytic activity. Several variables (pH, time, BrNAP concentration)were examined in an unsuccessful attempt to identify reaction conditions that gave selective binding of BrNAP to a single residue in cytochrome P-45Oc. These experiments did reveal, however, that the rate of inactivation and alkylation of cytochrome P-45Oc by BrNAP increased with increasing pH over the range pH 6.5 to 8.5 (results not shown). Antibody Inhibition Experiments-Antibody inhibition experiments were performed to test thepossibility that the 1015%catalytic activity remaining after cytochrome P-45Oc was alkylated with BrNAP was due to a contaminating isozyme in the cytochrome P-45Oc preparation. The catalytic activity of alkylated cytochrome P-45Oc wasdetermined with benzo[a] pyrene as substratein the presence and absence of monoclonal antibody C8 (1.5 mol of IgG/mol of cytochrome P-45Oc) (30). The monoclonal antibody inhibited >97% of the residual benzo[a]pyrene hydroxylase activity catalyzed by alkylated cytochrome P-45Oc. Similar results were obtained with 7ethoxycoumarin and zoxazolamine as substrates. Inasmuch as monoclonal antibody C8 reacts with a single peptide ( M , = 56,000) in “Westernblots” of rat liver microsomes (30), the antibody inhibition experiment demonstrated that a contaminating isozyme of cytochrome P-450 is not responsible for the 10-15% residual catalytic activity of BrNAP-treated cytochrome P-45Oc. Finally, the catalytic effectiveness of cyto-

Chemical Modification

of Rat Cytochrome P-45Oc

11483

Structure-ActivityRelationships-Severalcompounds chrome P-45Oc for benzo[a]pyrene metabolism ( Vmax)is >lofold higher than the next most active isozyme (2-7), and the structurally related to BrNAP were tested at two concentraresidual catalytic activity of the alkylated protein is still tions (10 and 50 ~ L Mfor) their ability to alkylate and inactivate , theresults are shown in Table higher than any otherknown rat cytochrome P-450 isozyme. cytochrome P-45Oc (1p ~ ) and The Effects of Detergents-Regardless of pH, incubation 11. The alkylation of cytochrome P-45Oc by these compounds time, and concentration of BrNAP, treatment of cytochrome was measured by their ability to block the subsequent covalent P-45Oc with BrNAP results in no more than 90% inhibition binding of [14C]BrNAP. As expected, 4-nitroacetophenone of catalytic activity toward benzo[a]pyrene. The residual cat- was inactive. Bromoacetic acid also failed to block the ability alytic activity of alkylated cytochrome P-450c7which is not of BrNAPto alkylate and inactivate cytochrome P-450c, due to a contaminant in the cytochrome P-45Oc preparation, whereas 2-bromoacetophenone (removal of the nitro group) has importantimplications regarding the natureof the amino was moderately effective in preventing the binding of [“C] acid residue(s) alkylated by BrNAP. If BrNAP alkylated each BrNAP. Although 2-(2-bromoacetyl)-naphthalenewas an efwas not. molecule of cytochrome P-45Oc and each molecule retained fective blocking agent, 9-[2-bromoacetyl]-anthracene 10-15% of its catalytic activity, the residue alkylated would Substitution of the bromoacetyl group of 2-bromoacetophennot, by definition (41, 42), be an essential amino acid for one with a phenyl group (2-bromo-2-phenylacetophenone) catalytic activity. Alternatively, if BrNAP alkylated only 85- caused a reduction of reactivity. Interestingly, 2-bromoac90% of the cytochrome P-45Oc molecules but each molecule etamido-4-nitrophenol, which reacts rapidly with a cysteine were completely inactivated, the residue alkylated would be residue in cytochrome P-450cam (47), failed to inactivate an essential amino acid. In this case, the observed residual cytochrome P-45Oc and to block the subsequent binding of activity would be catalyzed by 10-15% of the cytochrome P- BrNAP. 450c molecules that, by being in an aggregated state, were For most of the above-mentioned compounds (the firsteight inaccessible to alkylation by BrNAP. To try to distinguish listed in Table 11), there was good correspondence between between these two possibilities, cytochrome P-45Oc (1 ~ L M )the ability of each compound to inhibit the catalytic activity was treated with a 20-fold molar excess of BrNAP in the of cytochrome P-45Oc toward benzo[a]pyrene and to block presence of 0.5% sodium cholate, CHAPS, or octyl glucoside, the subsequent binding of BrNAP to cytochrome P-45Oc. i.e. detergents known to dissociate aggregates of cytochrome However, this relationship was not upheld by the last four P-450 (45, 46). After the detergents and excess BrNAP were compounds listed in Table 11. Treatment of cytochrome Premoved by dialysis, the catalytic activity of purified cyto- 450c with 2-(2-bromoacetyl)-fluoreneor 2-, 3-, or 9-(2-brochrome P-45Oc was measured with benzo[a]pyrene as sub- moacety1)-phenanthrene effectively blocked the subsequent strate. The results (not shown) indicated that, even under binding of BrNAP to cytochrome P-45Oc but inhibited the conditions when the aggregation state of cytochrome P-45Oc catalytic activity of cytochrome P-45Oc by only -60% (comis considerably reduced (45, 46), alkylation with BrNAP re- pared to -80% inhibition by BrNAP). The experiments with sults in only an -85% inactivation of cytochrome P-45Oc. the last four compounds listed in Table I1 were repeated, and These resultsstrongly suggest that each molecule of alkylated the results confirmed the ability of 2-(2-bromoacetyl)-flucytochrome P-45Oc retains -15% of its catalytic activity. orene andthe threebromoacetyl derivatives of phenanthrene to block the subsequent binding of BrNAP to cytochrome PPDS-Treated Cytochrome P450c 450c without inactivating cytochrome P-45Oc to the same 100 e e extent as BrNAP. 8 e 9””“---”-r””-. It is noteworthy that many of the compounds listed in BrNAP-Treated. PDS-Pretreated I1 have the potential to undergo substrate-like interTable Cytochrome P45Dc 80 I/’ I actions with cytochrome P-45Oc. We have shown previously I I that cytochrome P-45Oc metabolizes naphthalene andanthraI I cene at the 1,2-position and metabolizes phenanthrene at 60 - / I the 1,2-, 3,4- and 9,lO-positions (23, 48). Consequently, 2I I (2-bromoacetyl)-naphthalene, 9-(2-bromoacetyl)-anthracene, I I and 2-, 3-, and 9-(2-bromoacetyl)-phenanthreneare substrate 40 -/ I analogs that might be expected to position the bromoacetyl I group in the vicinity of the active site of cytochrome P-45Oc. The results in Table I1show that the structure of the substituent influences the ability of the bromoacetyl derivatives to alkylate cytochrome P-45Oc at the same sites(s) as BrNAP. This dependence on the structure of the alkylating OL I I I I 0 2 4 6 8 agent suggests that the residue(s) alkylated by BrNAP are MOLAR RATIO OF PDS TO CYTOCHROME P450c 1 1 I I not freely exposed on cytochrome P-450c, but are possibly COVALENT I BINDING OF ErNAP 1.53 -0.70 0.46 -0.35 -0.35 located in amicroenvironment that limits the access of certain (mol/mol CYTOCHROME1 compounds, such as thebromoacetyl derivative of anthracene. FIG. 2. Effect of pretreatment with the sulfhydryl reagent More importantly, however, compounds capable of binding to PDS on the inactivation and alkylation of cytochrome P-4SOc the same residue(s) as BrNAP do not always inactivate cytoby BrNAP. Cytochrome P-45012 (1 PM) was treated with a 2-, 4-, chrome P-45Oc to the same extent as BrNAP. Furthermore, 6-, or 8-fold molar excess of PDS for 30 min a t 22 “C. The reaction none of the compounds tested inhibited the catalytic activity by-product, 4-thiopyridone, and any excess PDS were removed by overnight dialysis against 2 X 2 liters of potassium phosphate buffer of cytochrome P-45Oc more than90%. Thedata provide (100 mM, pH 7.4) containing 20%glycerol a t 4 “C. The PDS-treated further evidence that theresidues alkylated by BrNAP do not cytochrome P-45Oc was either tested for catalytic activity or treated include an essential amino acid (ie. anamino acid that when with a 5-fold molar excess of [“CIBrNAP for 30 min at 22 “C. Covalent binding of BrNAP and catalytic activity of the BrNAP- alkylated completely inactivates the enzyme). The data sugtreated, PDS-pretreated cytochrome P-45Oc were determined as de- gest that thealkylation of cytochrome P-45Oc by BrNAP and scribed under “Experimental Procedures.” related compounds decreases its catalytic efficiency and that 1

2ol

-

11484

Chemical Modificationof Rat Cytochrome P - 4 5 0 ~ 0.2- A

- 100 -00 8

g

-0

v50

60

90 TIME (mid

120

180

L

Jn

%o

60

120

90 TIME (mid

180

FIG.3. HPLC profile of tryptic peptides from PDS-pretreated, [‘“ClBrNAP-alkylated cytochrome P-45Oc (upper) and [“ClBrNAP-alkylated protein (lower). Cytochrome P-45Oc and PDS-treated cytochrome P-45Oc were alkylated with [“C]BrNAP, dialyzed against ammonium bicarbonate (200 mM, pH 8.0), and digested with TPCK-trypsin. The tryptic peptides were resolved by reverse-phase HPLC, as described under “Experimental Procedures.” A 2 0 4 aliquot was counted from each fraction (fraction sizes ranged from 0.5-1.0 ml) and plotted for each chromatogram (uncorrected for volume differences between samples). TABLEI11 Microsequence analysis of BrNAP-labeled tryptic peptides Residue identified“ Cycle + + -

-

SCHEME I the extent to which the catalytic competency of cytochrome P-45Oc is compromised is dependent on the chemical structure of the alkylating agent. It is conceivable that some of the compounds listed in Table I1 do not alkylate cytochrome P450c but block the subsequent binding of BrNAP in a competitive manner. However, the inability of benzo[a]pyrene and othersubstrates to block the subsequent binding of BrNAPto cytochrome P-45Oc (see above) makes this an unlikely possibility. Alkylation with the Sulfhydryl Reagent, PDS-A cysteine residue(s), other than the heme-binding thiolate ligand, was a likely candidate for the site of alkylation in cytochrome P450c byBrNAP (43,44).The effect of pretreating cytochrome P-45Oc with the sulfhydryl reagent, PDS, on the subsequent binding of [14C]BrNAPwas examined in an attempt provide to further evidence that cysteine is the major aminoacid(s) alkylated by BrNAP.Pretreatment of cytochrome P-45Oc with as little as 2 molar eq of PDS significantly blocked the subsequent binding of BrNAP (Fig. 2).3The subsequent binding of BrNAP was still blocked after the PDS-treated cytochrome P-45Oc was dialyzed to remove 4-thiopyridone, the side product formed during the alkylation of sulfhydryl groups by PDS. This result established that the ability of PDS to block the subsequent binding of BrNAP to cytochrome P3Exposure of PDS-alkylated cytochrome P-45Oc to DTT was avoided to prevent reversal of the chemical modification (31,43).

BrNAP-3 BrNAP-2 BrNAP-1 pmol

1 2 3 4 5

6 7 8 9 10 11 12 13

ASP (68) Ile (79) Thr (14) ASP (30) Ser (15) Leu (36) Ile (34) Glu (15) His (19) (CYSI Gln (19) ASP (5) Arg (+)

Asp (158) Ile (306) Thr (45) Asp (112) Ser (39) Leu (101) Ile (79) Glu (63) His (46) (CYS) Gln (19) ASP (20) Arg (+I

Ser (39) Phe (83) Ser (15) Ile (28) Ala (33) Ser (4) ASP (12) Thr (+) Thr (+) Ala (+) Ser (+) Ser (+) (CY4

3520 5568 2560 Total CDm/DeDtide The amounts of BrNAP-1, -2, and -3 sequenced were 200, 350, and 200 pmol, respectively. Yields a t each cycle are given in pmol. A (+) indicates that the yield was too low to quantitate. No derivative was identified at cycle 10 for peptides BrNAP-1 and BrNAP-2, but cysteine is the known residue in this sequence (18).The amount of [14C]BrNAPincorporated per peptide is given as cpm/peptide. No cpm were recovered for the phenylthiohydantoin derivatives which were counted at each cycle. The low yields prevented identifications beyond cycle 13 for BrNAP-3. The occurrence of identical sequences for the separated peptides BrNAP-1 andBrNAP-2 may be due to the acid liability of the Cys-BrNAP adduct.

450c was not an artifact caused by the binding of BrNAP to 4-thiopyridone. In addition to blocking the binding of BrNAP, pretreatment of cytochrome P-45Oc with PDS also blocked the inactivation of cytochrome P-45Oc by BrNAP (Fig. 2). These results also

11485

Chemical Modification of Rat Cytochrome P-45Oc BrNAP-I

Alp Ik

Thr Asp Ser

Asp Ik

Thr Gly Ala

A l p Phe Ile

BrNAP-3

Leu Ik Clu His CYS Gln Asp Arg Leu Phe Lys His Ser Glu Asn

Asp Thr Tyr Leu Leu

Scr Phe Ser Ile Ala Scr Phc

-

-

Ser I k

Arq Mel Clu Lys Glu

Ser Asp Pro Thr Leu Ala

Ala Scr Asp Pro Thr Ser Val

Lys Arg Ser Val Clu Clu A r g Ile

-

P-4%

(233-295)

P-45Od

1281-292)

P-lmabk

(263-275)

Ser Ser CYS Tyr Leu Glu Glu His Val

Ser Lys

Ser Ser CYS Tyr Leu Glu Glu His Val Ser Lyr

Gln Glu Clu Ala Cln CYS - Leu Val Glu Glu

Leu Arq Lys

P-4%

(147-I681

P-45Od

(145-1661

P-4Wle

(139-1591

FIG. 4. Amino acid sequence of the majortryptic peptides alkylated by treatment of cytochrome P450c with BrNAP. The amino acid sequence of each radiolabeled cytochrome P-45Oc peptide was determined by automated Edman degradation (33). The tryptic peptides BrNAP-1 and -2 correspond to T-36, and BrNAP-3 corresponds to T-46 (18) in the unalkylated protein and are amino acids 283-295 and 147-168, respectively (16, 17). This numbering system and that of Yabusaki et al. (16) differs by 1 from that reported by Sogawa et al. (17) for the genomic sequence because these latter authors included the NH2-terminal methionine that is absent from purified cytochrome P-45Oc (16, 21). Corresponding amino acid sequences in cytochrome P-450d (19, 20) and in both cytochromes P-450b and P-450e (13-15) are shown for comparative purposes. Gaps are introduced to maximize homology.

for the unalkylated show that alkylation of cytochrome P-45Oc with PDS does quence of BrNAP-3 was the same as T-46 protein (18).Peptides BrNAP-1 and BrNAP-3 each contain not result in a loss of catalytic activity, as was previously observed by Kawalek et al. (31).One possible explanation for one cysteine residue (Cys-160 and Cys-292; Fig. 4). The identhe ability of PDS to bind to the same amino acid residue(s)tification of the residues alkylated by BrNAP was complicated by tendency of the adducts todecompose when the peptides in cytochrome P-45Oc as BrNAP without inactivating the Various enzyme is that the PDS adduct decomposes when the heme- were subjected toautomatedEdmandegradation. attempts to stabilize the adduct provedunsuccessful and, protein is reconstituted with NADPH-cytochrome P-450 reductase, lipid, and NADPH. To investigate this possibility, hence, the alkylated amino acid could not be identified diwe wished to determine whether 4-thiopyridone is released rectly. However, all of the amino acids except cysteine gave clear signals when BrNAP-1, -2, and -3 were subjected to from the PDS-modified enzyme upon incubation with NADPH,NADPH-cytochromeP-450reductase,and lipid. Edman degradation. These results, together with the ability Because of the highly polar nature of 4-thiopyridone (a zwit- of PDS to decrease the binding of BrNAP to cytochrome Pof Cysterionic resonance structure can be drawn), direct extraction 450c (Fig. 2 and 3), are consistent with the alkylation into organic solventswas not practical, and it was necessary 160 and Cys-292 in cytochromeP-45Oc by BrNAP. Theoverto trap this compoundby forming a less polar derivative. For all recovery and specific activities of the peptides show that this purpose, reaction with l-chloro-2,4-dinitrobenzene,the major and minor alkylated cysteines are Cys-292 and Cyswhich forms a highly absorbing (330 nm) product, was em- 160, respectively. In another alkylation experiment, the major ployed (Scheme I). radiolabeled peptides isolated were BrNAP-1 and BrNAP-2. Incubation of cytochrome P-45Oc (modified with 2 mol of Very little BrNAP-3 was detected (data not shown). This experiment further suggests that Cys-292 is the major alkylPDS/mol of protein)witheitherNADPHandNADPHated amino acid. cytochromeP-450reductaseorNADPHalonecausedthe rapid release of 1.6-1.9 mol of 4-thiopyridone/mol of cytoAlthough the results of both experiments provide strong chrome P-45Oc. Product identification was made as described evidence that BrNAP alkylates primarily Cys-292 in cytounder “Experimental Procedures.” Thus, the lack of effect of chrome P-450c, they do not estabIish whetheronly this resiPDS treatment on the enzymatic activity of cytochrome P-45Oc due is involved in catalytic inactivation of the enzyme. Comcan be accounted for by regeneration of the free enzyme upon parison of the aminoacid sequence of BrNAP-1 and BrNAPreaction of the PDS-modified protein with NADPH. 3 withthecorrespondingamino acidsequences incytoIdentification of the Alkylated Residues-To identify dichromes P-450d, P-450b, and P-450e (Fig. 4) reveals that, in rectly the amino acid residues alkylated by BrNAP, cytocontrast to Cys-160, thereisnocysteine residuein cytochrome P-45Oc (1 p M ) was treated with 5 molar eq of [“C] chromesP-450dorP-450b/e corresponding to Cys-292 in BrNAPfor 30 min a t room temperature, before orafter cytochrome P-450c, i.e. Cys-292 is substituted by a serine in pretreatment of the protein with PDS. Afterdialysis against cytochromeP-450d (16-20) and by glutamicacid in cytoammonium bicarbonate buffer, the alkylated cytochrome P- chromes P-450b and P-450e (13-15). In contrast to Cys-292, 450c preparations were digested with TPCK-treated trypsin Cys-160 is part of an amino acidsequence that is highly and the peptides separated by reverse-phase HPLC (18), as conserved between cytochromes P-45Oc and P-450d (16-20). detailed under “Experimental Procedures.” Three major and It is tempting to speculate, therefore, that the “uniqueness” several minor radiolabeled tryptic peptides were identified in of Cys-292 exlains why onlycytochrome P-45Oc suffersa the cytochrome P-45Oc preparation treated only with [“C] substantial loss of catalyticactivity when alkylated with BrNAP (Fig. 3). Pretreatment of cytochrome P-45Oc with BrNAP. When an attemptwas made to isolate [14C]BrNAPPDS, which decreased the subsequent bindingof [14C]BrNAP alkylated peptides from cytochrome P-450d, no major radiofrom 1.6 to 0.7 mol/mol of cytochrome P-450c,caused a active peptide was identified. The labeling pattern indicated marked decrease in the amount of radioactivity associated a broad distribution of nonspecific labeling including Cyswith the three major peptides but had little effect on the 158, the equivalent of Cys-160 in cytochrome P-45Oc (data subsequentbinding of [14C]BrNAP to the minorsites of not shown). This experiment strengthens the argument that alkylation (Fig.3).Microsequence analysis(Table 111) re- Cys-292 is the primary targetresidue for alkylation followed vealed thattheamino acid sequences of BrNAP-1and by catalytic inactivation of cytochrome P-45Oc. BrNAP-2 were identical (see footnote to Table 111) and that In summary, the results of the present study show that they corresponded to peptide T-36 previously sequenced from BrNAP alkylates each of 10 purified isozymes of rat liver unalkylatedcytochrome P-45Oc (18). Theamino acid se- microsomal cytochromeP-450 (P-450a-P-450j), as well as

11486

Chemical Modification of Rat Cytochrome P-45Oc

NADPH-cytochromeP-450reductase.Amongthe 10 isozymes of cytochromeP-450tested,cytochrome P-45Oc is unique in that alkylation with BrNAP causes a substantial loss of catalytic activity. The inactivation of cytochrome P450c by BrNAP involves primarily alkylation of Cys-292 and, to a lesser extent, Cys-160, neither of which is the putative heme-binding cysteine (Cys-460). The mechanism by which BrNAP inactivates cytochrome P-45Oc is the subject of the accompanying paperand involves an uncoupling of the catalytic cycle with the consequent formation of superoxide anion. Acknowledgment-We thank M. Floyd for her assistance in the preparation of this manuscript.

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Ryan, D. E., Iida, S., Wood, A. W., Thomas, P. E., Lieber, C. S., and Levin, W. (1984) J. Biol. Chem. 259, 1239-1250 Ryan, D. E., Dixon, R., Evans, R. H., Ramanathan, L., Thomas, P. E.,Wood, A. W., and Levin, W. (1984) Arch.Biochem. Biophys. 233,636-642 Ryan, D. E., Ramanathan, L., Iida, S., Thomas, P. E., Haniu, M., Shively, J. E., Lieber, C. S., and Levin, W. (1985) J. Biol. Chem. 260,6385-6394 Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1981) J. Biol. Chem. 256, 1044-1052 Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1983) J. Biol. Chem. 258,4590-4598 Parkinson, A., Safe, S. H., Robertson, L.W., Thomas, P. E., Rvan. D. E., Reik, L. M., and Levin, W. (1983) J. Biol. Chem. 258,'5967-5976 Botelho. L. H.. Rvan. D. E.. and Levin., W. (1979) . _ J. Biol. Chem. 254,5635-5640 ' Botelho, L. H., Ryan, D. E., Yuan, P.-M., Kutney, R., Shively, J. E., and Levin, W. (1982) Biochemistry 21,1152-1155 Yuan, P. M., Ryan, D. E., Levin, W., and Shively, J. E. (1983) Proc. Natl. Acud. Sci. U. S. A. 80, 1169-1173 Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci.U. S. A . 79,27932797 Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M., and FujiiKuriyama, Y. (1983) Proc. Natl. Acud. Sci. U. S. A . 80, 39583962 Yabusaki, Y., Shimizu, M., Murakami, H., Nakamura, K., Oeda, K., and Ohkawa, H.(1984) Nucleic Acids Res. 12,2929-2938 Sogawa,K., Gotoh, O., Kawajiri, K., and Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. U. S. A . 8 1 , 5066-5070 Haniu, M., Yuan, P.-M., Ryan, D. E., Levin, W., and Shively, J. E. (1984) Biochemistv 23,2478-2482 Kawajiri, K., Gotoh, O., Sogawa, K., Tagashira, Y., Muramatsu, M., and Fujii-Kuriyama, Y. (1984) Proc. Natl. Acud. Sci. U. S. A . 81, 1649-1653

20. Haniu, M., Ryan, D. E., Levin, W., and Shively, J. E. (1984) Proc. Natl. Acud. Sci. U. S. A . 8 1 , 4298-4301 21. Haniu, M., Ryan, D. E., Iida, S., Lieber, C. S., Levin, W., and Shively, J. E. (1984) Arch. Biochem. Biophys. 235, 304-311 22. Jerina, D. M., Michaud, D. P., Feldman, R. J., Armstrong, R. M., Vyas, K. P., Thakker, D. R., Yagi, H., Thomas, P. E., Ryan, D. E., and Levin, W. (1982) in Microsomes, Drug Oxidations and Drug Toxicity (Sato, R., and Kato, R., eds) pp. 195-201, Japan Scientific Societies Press, Tokyo 23. van Bladeren, P. J., Vyas,K.P., Sayer, J. M., Ryan, D. E., Thomas, P. E., Levin, W., and Jerina, D.M. (1984) J. Biol. Chem. 259,8966-8973 24. Levin, W., Wood, A. W., Chang, R., Ryan, D., Thomas, P., Yagi, H., Thakker, D., Vyas, K., Boyd, C.,Chu, S.-Y., Conney, A. H., and Jerina, D. M. (1982) Drug Metab. Rev. 13, 555-580 25. DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 2 5 3 , 2932-2939 26. May, E. J., and Mosettig, E. (1948) J. Am. Chem. SOC.7 0 , 686688 27. Dignam, J. D., and Strobel, H. W. (1975) Biochem. Biophys. Res. Commun. 63,845-852 28. Yasukochi, Y., and Masters, B. S. S. (1976) J. Biol. Chem. 2 5 1 , 5337-5344 29. Phillips, A. H., and Langdon, R. G. (1962) J. Biol. Chem. 237, 2652-2660 30. Thomas, P. E., Reik, L. M., Ryan, D. E., and Levin, W. (1984) J. Biol. Chem. 259,3890-3899 31. Kawalek, J. C., Levin, W., Ryan, D., and Lu, A. H. H. (1977) Arch. Biochem. Biophys. 183,732-741 32. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239,2379-2385 33. Hawke, B., Harris, D., and Shively, J. E. (1985) Anal. Biochem. 147, 315-330 34. Hawke. C.D.. Yuan. P.-M.. and Shivelv. . . Anal. " . J. E. (1982) Biochem. 120,3021311 35. Reik. L. M.. Levin. W.. Rvan. D. E.. and Thomas. P. E. (1982) . , J. Biol. Chek. 2 5 7 , 3950:3957 ' 36. Masters, B. S. S., and Okita, R. T. (1980) P h a r m o l . Ther. 9, 227-244 37. White, R. E., and Coon, M. J. (1980) Annu. Reu. Biochem. 49, 315-356 38. Ullrich, V. (1979) Top. Curr. Chem. 83,67-104 39. Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, s.,and Omura, T. (1984) Proc. Natl. Acud. Sci. U. S. A . 81,4647-4651 40. Black, S. D., and Coon, M. J. (1985) Biochem.Biophys.Res. Commun. 128,82-89 41. Kitz, R., and Wilson, I. B. (1962) J. Biol. Chem. 237, 3245-3249 42. Plapp, B. V. (1982) Methods Enzymol. 87,469-495 43. Lundblad, R. L., and Noyes, C. M. (1984) Chemical Reagents for Protein Modification. Vol. I.. "DD. 55-94. CRC Press,. Inc.,, Boca Raton, FL 44. Kyger, E. M., and Franson, R. C. (1984) Biochim. Biophys. Acta 794,96-103 45. Hielmeland. L. M. (1980) . . Proc.Natl. Acud. Sci. U. S. A . 77. "6368-6370 46. Dean, W. L., and Gray, R.D. (1982) Biochem.Biophys.Res. Commun. 107,265-277 47. Haniu. M.. Yasunobu. K. T.. and Gunsalus. I. C. (1982) . . Biochem. Bio&ys.'Res. Commun. 107,1075-1081' 48. Nordquist, M., Thakker, D. R., Vyas, K. P., Yagi, H., Levin, W., Ryan, D. E., Thomas, P. E., Conney, A. H., and Jerina, D. M. (1981) Mol. Phurmucol. 1 9 , 168-178 '