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Chemical Modification of Microsomal Cytochrome P450: Role of Lysyl Residues in Hydroxylation Activity

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Chemical Modification of Microsomal Cytochrome P450: Role of Lysyl Residues in Hydroxylation Activity View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 161, number 2 J?EBS 0813 September 1983 Chemical modification of microsomal cytochrome P450: role of lysyl residues in hydroxylation activity Barbara C. Kunz and Christoph Richter* Eidgeniissische Technische Hochschule, Laboratorium fiir Biochemie I, Universitiitstrasse 16, CH-8092 Zurich, Switzerland Received 4 August 1983 Cytochrome P450 purified from phenobarbital-induced rat liver microsomes was acetylakd at 3 lysyl residues. When reconstituted with purified NADPH-cytochrome P450 reductase, the modified cytochrome showed full activity and substrate-induced spectral changes with d-benzphetamine. With 7-ethoxycoumar- in, neither enzymic activity nor binding was detected. It is concluded that the positively charged lysine residues of cytochrome P450 are important for metabolism of ‘I-ethoxycoumarin by cytochrome P450. Microsomal monoxygenase Cytochrome P450 activity Acetylation Reconstitution 1. INTRODUCTION stoichiometry (20-30 cytochromes/recfuctase) raises questions as to the mechanism of electron Cytochrome P450 and NADPH-cytochrome transfer from the reductase to the cytochrome and P450 reductase are key enzymes of the hepatic the functional interactions of the proteins in the microsomal monooxygenase system, catalyzing the monooxygenase system. oxidative metabolism of endogenous substrates Some progress has been made in our under- and many xenobiotics [l-3]. The reductase standing of the structure of cytochrome P450. (Mr - 78000) is anchored to the membrane via a Amino acid sequences of several species are now small (M- 6000) hydrophobic segment [4,5]. The available [7,8]. In addition the dimension of the large hydrophilic part protrudes from the mem- heme pocket has been partially characterized by brane into the cytoplasmic space and accepts heme alkylation studies [9, lo]. electrons from NADPH. Cytochrome P450 By combining physical, biochemical and im- (M,-SOOOO), on the other hand, is deeply imbed- munological techniques we demonstrated the for- ded in the membrane [6]. The arrangement of the mation of heterodimeric complexes between two enzymes in the membrane and their odd cytochrome P450 and its reductase co-reconstitu- ted into liposomal vesicles and capable of substrate hydroxylation [ 1 l-131. We are now chemically * To whom correspondence should be addressed modifying specific sites of the proteins to improve our understanding of the functional interactions of Abbreviations: PC, phosphatidylcholine; PE, phospha- tidylethanolamine; PS, phosphatidylserine; SDS- the proteins. Here, we show that limited acetyla- PAGE, sodium dodecylsulfate-polyacrylamide gel elec- tion of lysyl residues of cytochrome P450 leads to trophoresis; EDTA, ethylene diaminetetraacetate; a complete loss of ethoxycoumarin dealkylation HEPES, 4-(2-hydroxyethyl)-I-piperazineethane sulfonic activity whereas the dealkylation of benzphet- acid amine is not affected. Published by Elsevier Science Publishers B. V. 00145793/83/$3.00 0 1983 Federation of European Biochemical Societies 311 Volume 161, number 2 FEBS LETTERS September 1983 2. MATERIALS AND METHODS groups [ 191 was applied to cytochrome P450. One volume of cytochrome P450 (26 PM), pre- Phospholipids [egg phosphatidylcholine (PC), equilibrated with 50 mM HEPES (pH 8.0, 2O@i’o egg phosphatidylethanolamine (PE), bovine spinal glycerol), was mixed with half this volume of a cord phosphatidylserine (PS), all grade I] were freshly prepared, saturated sodium acetate solu- purchased from Lipid Products (South Nutfield) tion, containing 20% glycerol and [3H]acetic and stored at - 20°C. Dilauroylphosphatidyl- anhydride (10 mM, 30 &i/ml). The solution was choline was from Sigma (St. Louis, MO). [‘HI- stirred at room temperature. After 10 min the same Acetic anhydride (500 mCi/mmol) was from the amount of the above-mentioned sodium acetate Radiochemical Centre (Amersham). d-Benzphet- acetic anhydride solution was added. Stirring was amine was from Upjohn. 7-Ethoxycoumarin was continued for 20 min. Cytochrome P450 in vesicles prepared as in [14]. All other reagents were of the was acetylated by the same procedure. highest grade available. 2.4. Determination of the acetylated amino acids 2.1. Analytical procedures 3H was measured with an Isocap 300 liquid scin- All spectral analyses were performed at 25°C us- tillation counter, after separation of the protein by ing a Cary 219 spectrophotometer. Cytochrome SDS-PAGE. The gel containing cytochrome P450 P450 concentration was determined as in [ 151. Pro- was sliced and dissociated in 500 ~1 hydrogen tein concentration was measured by the Lowry peroxide (30%) at 60°C overnight, prior to adding assay [16] using bovine serum albumin as a stan- the scintillation liquid. The nature of the acetyl- dard. SDS-PAGE was performed according to ated residues was affirmed by amino acid analysis Laemmli [17]. after dansylation [20] of the protein. Isolation of cytochrome P450, NADPH- cytochrome P450 reductase and the preparation of 2.5. Enzyme assays proteoliposomes (PC/PE/PS, 10: 5 : 1, by wt) was Ethoxycoumarin 0-de-ethylase activity was perfofmed as in [12]. Either protein showed a measured as in [14] except that cytochrome P450 single band when analyzed on SDS-PAGE. Addi- either in vesicles or in solution in the presence of tional evidence that we have only a single species of a NADPH regenerating system (glucose 6-phos- cytochrome P450 in our preparation is supported phate (5 mM), glucose 6-phosphate dehydrogenase by its similar catalytic activity and substrate (2.5 units)) was used instead of microsomes. Benz- specificity to that of cytochrome P450 PB-B phetamine hydroxylation was determined as in described in [18]. For the enzyme assays, [ 121. Substrate-induced difference spectra in the acetylated cytochrome P450 containing vesicles Soret region were recorded in tandem cuvettes with were dissociated by cholate. NADPH-cytochrome vesicles in 100 mM KPi (pH 7.4, 20% glycerol). P450 reductase was added and vesicles were form- ed again by the cholate-dialysis techniques. 3. RESULTS 2.2. Reconstitution in dilauroylphosphatidyl- choline Specific acetylation of lysyl residues can be Cytochrome P450 and NADPH-cytochrome P450 achieved with acetic anhydride in the presence of reductase were mixed with freshly sonicated sodium acetate [19]. When dimethyl sulfoxide was dilauroylphosphatidylcholine (0.2% (w/w) in 0.1 used as solvent of acetic anhydride to prevent its mM EDTA) to give protein/lipid = 1: 10 (w/w). rapid hydrolysis cytochrome P450 proved un- The samples were incubated for 10 min at room stable. At 5°C and with a 400-fold excess of acetic temperature. In all reconstitution experiments the anhydride over cytochrome P450 in aqueous buf- molar ratio of cytochrome P450 to reductase was fer no significant heme loss (< 5%) or denatura- 5:l. tion of the cytochrome as judged by heme *CO spectroscopy was detected. The extent of acetyla- 2.3. Acetyiation of cytochrome P4.50 tion was reproducible under these conditions. A method for the acetylation of e-lysyl-amino Fig.1. shows the protein pattern and distribution 312 Volume 161, number 2 FEBS LETTERS September 1983 lysis and amino acid analysis as in [21] (not shown). Acetylation of cytochrome P450 in solu- tion (i.e., in the presence of dilauroylphosphatidyl- choline) gave similar results. Acetylation of phos- pholipids was not detected. Acetylated cytochrome P450 reconstituted into liposomes or in solution showed 110% f 50% (n = 12) of the activity of the native protein with benzphetamine as substrate for hydroxylation. Within a single set of experiments reproducibility of these activity measurements was good ( f 10%). u l dpm Large variations were observed however between 1000 2000 3000 different sets of experiments, with values ranging from 30-194 nmol formaldehyde formed. min- * . Fig.1. Protein pattern and distribution of radioactivity nmol cytochrome P450 - ‘. The dealkylation rate of acetylated cytochrome P450. lo-15% SDS-PAGE of ethoxycoumarin catalyzed by native cytochrome was performed as in 1171; cytochrome P450 was P450 reconstituted into liposomes or in solution acetylated with [‘HIacetic anhydride as in section 2. Each lane was loaded with 0.12 nmol cytochrome P450. was 250+ 26 pmol (n = 8) min- ’ . nmol cyto- chrome P450- ‘. No dealkylation of this substrate by acetylated cytochrome P450 was observed. The presence of poly(lysine) in amounts equimolar to of radioactivity of acetylated cytochrome P450 cytochrome P450 completely inhibited the analyzed on SDS-PAGE. From these experi- dealkylation of ethoxycoumarin whereas this ments, 2.8 +0.5 (n = 23) acetyl residues were polycation did not alter the hydroxylation of calculated to be bound per one cytochrome mole- benzphetamine. cule. Precipitation of the modified protein by acid Substrate binding studies of native or acetylated and subsequent filtration followed by liquid scin- cytochrome P450 reconstituted into liposomes or tillation indicated attachment of 3.0 f 0.2 (n = 3) in solution showed typical type I spectra with benz- acetyl groups to cytochrome P450. The specificity phetamine (fig.aa,b) while ethoxycoumarin induc- of the modification and the number of lysyl ed spectral changes only in the native (fig.2c) but residues modified was confirmed by acid hydro- not in the acetylated cytochrome. 350nm 420nm 5OOnm 420nm 500nm 4201~1 5OOm-l Fig.2. Substrate-induced difference spectra of cytochrome P450 reconstituted in liposomes:
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