[5]
P H O T O A F F I N I T Y PROBE OF C Y T O C H R O M E S P 4 5 0
57
[5] A z i d o w a r f a r i n as P h o t o a f f i n i t y P r o b e of Cytochromes P450 B y LAURENCE S. KAMINSKY, DEBORAH DUNBAR, a n d WILLIAM LAWSON
Introduction The extremely broad substrate specificity of the hepatic microsomal monooxygenase system is primarily a consequence of the multiplicity of the terminal oxygenase, cytochrome P450, although many of the individual cytochromes P450 also catalyze the metabolism of a number of substrates) '2 Since oxygen activation and substrate hydroxylation mechanisms are apparently common to all forms of cytochrome P450,3 the basis for the substrate specificity differences between forms of cytochrome P450 must be sought in the substrate binding site amino acid residues, and in their role of aligning the substrate for reaction with the activated oxygen. However, notwithstanding the availability of the primary structures of numerous cytochromes P450, there is a paucity of data on the identity of the amino acid residues which comprise the substrate binding site. The factors controlling cytochrome P450 substrate specificities are still unclear, and, in the absence of three-dimensional X-ray crystallographic data for mammalian cytochromes P450, labeling techniques for identifying substrate binding sites are the most likely to resolve the basis for substrate specificity. Photoaffinity labeling has the potential to be one of the best approaches for labeling cytochrome P450 substrate binding site amino acids, thus leading to their identification. The technique involves use of a substrate modified with a photoactivatable substituent, which specifically binds to the substrate binding site of an enzyme, is photoactivated in situ, and binds covalently to the substrate binding site. ¢-6 Aryl azides, which on I F. P. Guengerich, G. A. Dannan, S. T. Wright, M. V. Martin, and L. S. Kaminsky, Biochemistry 21, 6019 (1982). 2 C. S. Yang and A. Y. H. Lu, in "Mammalian Cytochromes P450" (F. P. Guengerich, ed.), p. 2. CRC Press, Boca Raton, Florida, 1987. 3 p. R. Ortiz de Montellano, in "Cytochrome P450 Structure, Mechanism, and Biochemistry" (P. R. Ortiz de Montellano, ed.), p. 217. Plenum, New York, 1986. 4 V. Choudhury and F. H. Westheimer, Annu. Reo. Biochem. 48, 293 (1979). 5 H. Bayley and J. R. Knowles, this series, Vol. 46, p. 69. 6 R. Lundblad and C. M. Noyes, in "CRC Chemical Reagents for Protein Modification, II," p. 141. CRC Press, Boca Raton, Florida, 1984.
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
58
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[5]
photoactivation yield nitrenes capable of inserting into carbon-hydrogen, oxygen-hydrogen, and nitrogen-hydrogen bonds, 5 are potentially useful for broadly labeling the substrate binding site residues of cytochromes P450, which are likely to have more than a single amino acid residue interacting with substrates. The mechanism of action of cytochrome P450 lends itself to a substrate-based photoaffinity probe of substrate binding sites, since substrates bind to the oxidized enzymes. Thus, binding of the probe to the cytochrome can occur in the absence of electron donors or reductase, and thus without undergoing metabolism. There are several potential limitations of photoaffinity probes for labeling substrate binding site residues of cytochromes P450. (1) The photoactivated probe could insert nonspecifically into nonsubstrate site amino acids. Such insertions could arise directly from photoactivated azidowarfarin in solution or by migration of a photoactivated substrate site-bound probe, and they could be diminished or prevented by the use of scavengers in solution or by conducting the photoactivation at 77 K. 7 (2) Modification of a substrate with a photoactivatable group could prevent its binding to the substrate binding site of the cytochrome P450. In the current studies this apparently occurred with one cytochrome P450 but not another, but use of a substrate with multiple sites for azido group substitution can alleviate this problem. The ultimate identification of substrate binding site amino acids is dependent on identification of those residues covalently bound by photoactivated probe. (3) During the protein sequencing required for bound residue identification, the bound label could be removed, or the label could interfere with the sequencing process. The former case has never been reported and is unlikely, particularly with photoactivated azido compounds; the latter is not a problem when cytochromes P450 of known sequences are used since the missing residue in a sequence will actually permit its identification by comparison of the partial sequence of the peptide with that of the protein. (R)-Warfarin and (S)-warfarin are potentially useful substrates for modification to photoaffinity labels, based on our results that these substrates are stereo- and regioselectively metabolized to dehydro-, 4'-, 6-, 7-, 8-, or 10-hydroxywarfarins by many forms of cytochrome P450. 8 Modification of (R)- and (S)-warfarin by substitution with azido moieties at various sites should provide a number of photoaffinity probes capable, as a group, of broadly labeling the substrate binding sites of a number of forms of
7 K. Kanakarajan, R. Goodrich, M. J. T. Young, S. Soundararagan, and M. S. Platz, J. Am. Chem. Soc. 110, 6536 (1988). 8 L. S. Kaminsky, M. J. Fas¢o, and F. P. Guengerich, this series, Vol. 74, p. 262.
[5]
PHOTOAFFINITY PROBE OF CYTOCHROMES P450
59
cytochrome P450. Here experimental details concerning the first of these probes synthesized, 4'-azidowarfarin, are provided. The photoaffinity probe, 4'-azidowarfarin, is labeled with tritium. The proposed reaction pathway involves the synthesis of dehydro-4'-nitrowarfarin by treatment of 4'-nitrowarfarin with cuprous chloride in pyridine, 8 formation of its ketal by treatment with methanol, and then reduction with NaBT4 to produce tritiated 4'-aminowarfarin methyl ketal. In the final step, tritiated 4'-azidowarfarin is synthesized by the method described below. The binding sites of the labeled photoaffinity probe on cytochrome P450 is determined by limited proteolysis of the protein, peptide mapping using high-performance liquid chromatography (HPLC) with a radioactive flow detector, isolation of labeled peptides using HPLC, and finally sequencing of labeled peptides to determine which specific amino acid residues are bound by labeled probe. Preparation of 4'-Azidowarfarin Racemic 4'-nitrowarfarin is prepared by modifications of the method of Bush and Tragcr. 9 4-Hydroxycoumarin (16.2 g, 0.1 M) and p-nitrobenzalacetone (19.8 g, 0.1 M) arc refluxed in methanol (810 ml) for 6 days. The solvent is removed in v a c u o and the residue dissolved in acetone (1215 ml) and I0 M HC1 (405 ml) and stirred at 37° for 24 hr. The solvent is removed in v a c u o , and the resultant solid is filtered, washed with cold 1 M HCI (3 x 50 ml), and air dried for 4 hr. The product is stirred in 90 mM NaOH (400 n-d) for 1.5 hr, and the solution is filtered and then treated with 6 M HCI (55 ml). The mixture is cooled in ice, and the precipitate is filtered and washed with 1 M HCI (3 × 50 ml). The product is recrystallized from hot acetone (500 ml) and water (300 ml) to yield yellow crystals (26.9 g, 77%), mp 191°-193 °. 4'-Azidowarfarin is prepared by modification of a published method. ~0 Racemic 4'-nitrowarfarin (2.0 g, 5.9 mM) is hydrogenated in the presence of 10% palladium on charcoal (0.2 g) in ethyl acetate (200 ml) for 24 hr at 51 lb/in. 2 and room temperature. The mixture is filtered and the volume of solvent reduced to 80 ml in v a c u o . The solution is cooled in ice and bubbled with HCI gas until the solution is slightly acidic (test with pH paper). Dry ether (100 ml) is added to precipitate the product, which is then filtered (2.15 g, 100%). The 4'-aminowarfarin hydrochloride (1.02 g, 2.83 raM) is dissolved in 2 M HCI (50 ml) at room temperature; the solution is cooled in ice, stirred, and a cold solution of NaNO 2 (0.283 g/ml water) is added 9 E. Bush and W. F. Trager, J. Pharm. Sci. 72, 830 (1983). l0 S. AImeda, D. H. Bing, R. Laura, and P. A. Friedman, Biochemistry 70, 3731 (1981).
60
MUTAGENESIS, MODIFICATION,PROTEIN STRUCTURE
[5]
dropwise. The solution is stirred for 10 min, and 2 M HCI (50 ml) is added. The solution is stirred for a further 30 min, then filtered; 8 M urea (0.8 ml) is added, and the solution is filtered again. NaN 3 (0.184 g/ml water) is added dropwise to the solution with stirring, which is continued for 1.5 hr at 0°. The resultant precipitate, which is 4'-azidowarfarin, is filtered (0.70 g, 70%), mp 850-90 °. The compound is stored in the dark, but even in solution it is stable to the ambient light in a fluorescent lamp-lighted laboratory for several days. Photoactivation of 4'-Azidowarfarin It has been demonstrated 7 that photoaffinity labeling at low temperature, 77 K, markedly improves thc yield of covalent attachment between the photoactivatcd azido compound (nitrcnc)and the targct enzyme. Photolysis at 77 K also results in a more simple reaction pathway. 7 Studies to optimizc the photoactivation of 4'-azidowarfarin usc an H P L C assay to assess the extent and nature of the outcome of photoactivation of 4'azidowarfarin. The H P L C method used is that developed for the analysis of warfarin and its mctabolitcs and has bccn described in detail,s Photolytic reactions are conducted in plastic petri dishcs (5.5 c m in diamcter), which arc floated on the surface of liquid nitrogen maintained in a shallow Dewar flask. The solution to bc irradiated is spread across the surface of the dish, which is kept in contact with the liquid nitrogen for 5 rainprior to irradiation.Irradiationis performed with a Model U V G L 25 Mincralight lamp (San Gabriel, CA), which is laid across thc top of thc pctri dish. Photolysis of a Tfis-HCI buffcrcd solution of 4'azidowarfarin (40 ~ M ) at room temperature and 366 or 254 n m yields products which on H P L C yield major peaks at retention times relativeto that of 4'-azidowarfarin of 0.37 and 0.54 and a minor peak at 0.87. Photolysis at 77 K yields a different product pattern, with the predominant product at a relativeretention time of 0.87 and a more minor product at 0.54. The 4'-azidowarfarin at 40 ftM is completely photoactivated after a 20-scc cxposurc to 254 n m light or five l-rainexposures to 366 n m light,as assessed by the disappearance of thc substrate, and the rate of its disappearance is independent of the temperature of the photolyzcd solution. Since photolysis of 4'-azidowarfafin at 254 n m is more efficient,studies with purified cytochromcs P450 arc conducted at this wavelength. However, in microsomal preparations 366 n m light is more efficientat photoactivating 4'-azidowarfarin and is thus preferentially used in photoaffinity labeling of microsomal-bound cytochromes P450. T w o criteriamust bc satisfiedin selecting a scavenger for photoactivatcd 4'-azidowarfarin. The potential scavcngcr should interact with and
[5]
PHOTOAFFINITY
0.4
I
PROBE
I
OF CYTOCHROMES I
F
i
P450 i
61 I
c"
E
o.s
0
/
v
J
O_
o
-.-t.
0.2
-5
E C w
i-.
P H O T O A F F I N I T Y PROBE OF C Y T O C H R O M E S P 4 5 0
57
[5] A z i d o w a r f a r i n as P h o t o a f f i n i t y P r o b e of Cytochromes P450 B y LAURENCE S. KAMINSKY, DEBORAH DUNBAR, a n d WILLIAM LAWSON
Introduction The extremely broad substrate specificity of the hepatic microsomal monooxygenase system is primarily a consequence of the multiplicity of the terminal oxygenase, cytochrome P450, although many of the individual cytochromes P450 also catalyze the metabolism of a number of substrates) '2 Since oxygen activation and substrate hydroxylation mechanisms are apparently common to all forms of cytochrome P450,3 the basis for the substrate specificity differences between forms of cytochrome P450 must be sought in the substrate binding site amino acid residues, and in their role of aligning the substrate for reaction with the activated oxygen. However, notwithstanding the availability of the primary structures of numerous cytochromes P450, there is a paucity of data on the identity of the amino acid residues which comprise the substrate binding site. The factors controlling cytochrome P450 substrate specificities are still unclear, and, in the absence of three-dimensional X-ray crystallographic data for mammalian cytochromes P450, labeling techniques for identifying substrate binding sites are the most likely to resolve the basis for substrate specificity. Photoaffinity labeling has the potential to be one of the best approaches for labeling cytochrome P450 substrate binding site amino acids, thus leading to their identification. The technique involves use of a substrate modified with a photoactivatable substituent, which specifically binds to the substrate binding site of an enzyme, is photoactivated in situ, and binds covalently to the substrate binding site. ¢-6 Aryl azides, which on I F. P. Guengerich, G. A. Dannan, S. T. Wright, M. V. Martin, and L. S. Kaminsky, Biochemistry 21, 6019 (1982). 2 C. S. Yang and A. Y. H. Lu, in "Mammalian Cytochromes P450" (F. P. Guengerich, ed.), p. 2. CRC Press, Boca Raton, Florida, 1987. 3 p. R. Ortiz de Montellano, in "Cytochrome P450 Structure, Mechanism, and Biochemistry" (P. R. Ortiz de Montellano, ed.), p. 217. Plenum, New York, 1986. 4 V. Choudhury and F. H. Westheimer, Annu. Reo. Biochem. 48, 293 (1979). 5 H. Bayley and J. R. Knowles, this series, Vol. 46, p. 69. 6 R. Lundblad and C. M. Noyes, in "CRC Chemical Reagents for Protein Modification, II," p. 141. CRC Press, Boca Raton, Florida, 1984.
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
58
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[5]
photoactivation yield nitrenes capable of inserting into carbon-hydrogen, oxygen-hydrogen, and nitrogen-hydrogen bonds, 5 are potentially useful for broadly labeling the substrate binding site residues of cytochromes P450, which are likely to have more than a single amino acid residue interacting with substrates. The mechanism of action of cytochrome P450 lends itself to a substrate-based photoaffinity probe of substrate binding sites, since substrates bind to the oxidized enzymes. Thus, binding of the probe to the cytochrome can occur in the absence of electron donors or reductase, and thus without undergoing metabolism. There are several potential limitations of photoaffinity probes for labeling substrate binding site residues of cytochromes P450. (1) The photoactivated probe could insert nonspecifically into nonsubstrate site amino acids. Such insertions could arise directly from photoactivated azidowarfarin in solution or by migration of a photoactivated substrate site-bound probe, and they could be diminished or prevented by the use of scavengers in solution or by conducting the photoactivation at 77 K. 7 (2) Modification of a substrate with a photoactivatable group could prevent its binding to the substrate binding site of the cytochrome P450. In the current studies this apparently occurred with one cytochrome P450 but not another, but use of a substrate with multiple sites for azido group substitution can alleviate this problem. The ultimate identification of substrate binding site amino acids is dependent on identification of those residues covalently bound by photoactivated probe. (3) During the protein sequencing required for bound residue identification, the bound label could be removed, or the label could interfere with the sequencing process. The former case has never been reported and is unlikely, particularly with photoactivated azido compounds; the latter is not a problem when cytochromes P450 of known sequences are used since the missing residue in a sequence will actually permit its identification by comparison of the partial sequence of the peptide with that of the protein. (R)-Warfarin and (S)-warfarin are potentially useful substrates for modification to photoaffinity labels, based on our results that these substrates are stereo- and regioselectively metabolized to dehydro-, 4'-, 6-, 7-, 8-, or 10-hydroxywarfarins by many forms of cytochrome P450. 8 Modification of (R)- and (S)-warfarin by substitution with azido moieties at various sites should provide a number of photoaffinity probes capable, as a group, of broadly labeling the substrate binding sites of a number of forms of
7 K. Kanakarajan, R. Goodrich, M. J. T. Young, S. Soundararagan, and M. S. Platz, J. Am. Chem. Soc. 110, 6536 (1988). 8 L. S. Kaminsky, M. J. Fas¢o, and F. P. Guengerich, this series, Vol. 74, p. 262.
[5]
PHOTOAFFINITY PROBE OF CYTOCHROMES P450
59
cytochrome P450. Here experimental details concerning the first of these probes synthesized, 4'-azidowarfarin, are provided. The photoaffinity probe, 4'-azidowarfarin, is labeled with tritium. The proposed reaction pathway involves the synthesis of dehydro-4'-nitrowarfarin by treatment of 4'-nitrowarfarin with cuprous chloride in pyridine, 8 formation of its ketal by treatment with methanol, and then reduction with NaBT4 to produce tritiated 4'-aminowarfarin methyl ketal. In the final step, tritiated 4'-azidowarfarin is synthesized by the method described below. The binding sites of the labeled photoaffinity probe on cytochrome P450 is determined by limited proteolysis of the protein, peptide mapping using high-performance liquid chromatography (HPLC) with a radioactive flow detector, isolation of labeled peptides using HPLC, and finally sequencing of labeled peptides to determine which specific amino acid residues are bound by labeled probe. Preparation of 4'-Azidowarfarin Racemic 4'-nitrowarfarin is prepared by modifications of the method of Bush and Tragcr. 9 4-Hydroxycoumarin (16.2 g, 0.1 M) and p-nitrobenzalacetone (19.8 g, 0.1 M) arc refluxed in methanol (810 ml) for 6 days. The solvent is removed in v a c u o and the residue dissolved in acetone (1215 ml) and I0 M HC1 (405 ml) and stirred at 37° for 24 hr. The solvent is removed in v a c u o , and the resultant solid is filtered, washed with cold 1 M HCI (3 x 50 ml), and air dried for 4 hr. The product is stirred in 90 mM NaOH (400 n-d) for 1.5 hr, and the solution is filtered and then treated with 6 M HCI (55 ml). The mixture is cooled in ice, and the precipitate is filtered and washed with 1 M HCI (3 × 50 ml). The product is recrystallized from hot acetone (500 ml) and water (300 ml) to yield yellow crystals (26.9 g, 77%), mp 191°-193 °. 4'-Azidowarfarin is prepared by modification of a published method. ~0 Racemic 4'-nitrowarfarin (2.0 g, 5.9 mM) is hydrogenated in the presence of 10% palladium on charcoal (0.2 g) in ethyl acetate (200 ml) for 24 hr at 51 lb/in. 2 and room temperature. The mixture is filtered and the volume of solvent reduced to 80 ml in v a c u o . The solution is cooled in ice and bubbled with HCI gas until the solution is slightly acidic (test with pH paper). Dry ether (100 ml) is added to precipitate the product, which is then filtered (2.15 g, 100%). The 4'-aminowarfarin hydrochloride (1.02 g, 2.83 raM) is dissolved in 2 M HCI (50 ml) at room temperature; the solution is cooled in ice, stirred, and a cold solution of NaNO 2 (0.283 g/ml water) is added 9 E. Bush and W. F. Trager, J. Pharm. Sci. 72, 830 (1983). l0 S. AImeda, D. H. Bing, R. Laura, and P. A. Friedman, Biochemistry 70, 3731 (1981).
60
MUTAGENESIS, MODIFICATION,PROTEIN STRUCTURE
[5]
dropwise. The solution is stirred for 10 min, and 2 M HCI (50 ml) is added. The solution is stirred for a further 30 min, then filtered; 8 M urea (0.8 ml) is added, and the solution is filtered again. NaN 3 (0.184 g/ml water) is added dropwise to the solution with stirring, which is continued for 1.5 hr at 0°. The resultant precipitate, which is 4'-azidowarfarin, is filtered (0.70 g, 70%), mp 850-90 °. The compound is stored in the dark, but even in solution it is stable to the ambient light in a fluorescent lamp-lighted laboratory for several days. Photoactivation of 4'-Azidowarfarin It has been demonstrated 7 that photoaffinity labeling at low temperature, 77 K, markedly improves thc yield of covalent attachment between the photoactivatcd azido compound (nitrcnc)and the targct enzyme. Photolysis at 77 K also results in a more simple reaction pathway. 7 Studies to optimizc the photoactivation of 4'-azidowarfarin usc an H P L C assay to assess the extent and nature of the outcome of photoactivation of 4'azidowarfarin. The H P L C method used is that developed for the analysis of warfarin and its mctabolitcs and has bccn described in detail,s Photolytic reactions are conducted in plastic petri dishcs (5.5 c m in diamcter), which arc floated on the surface of liquid nitrogen maintained in a shallow Dewar flask. The solution to bc irradiated is spread across the surface of the dish, which is kept in contact with the liquid nitrogen for 5 rainprior to irradiation.Irradiationis performed with a Model U V G L 25 Mincralight lamp (San Gabriel, CA), which is laid across thc top of thc pctri dish. Photolysis of a Tfis-HCI buffcrcd solution of 4'azidowarfarin (40 ~ M ) at room temperature and 366 or 254 n m yields products which on H P L C yield major peaks at retention times relativeto that of 4'-azidowarfarin of 0.37 and 0.54 and a minor peak at 0.87. Photolysis at 77 K yields a different product pattern, with the predominant product at a relativeretention time of 0.87 and a more minor product at 0.54. The 4'-azidowarfarin at 40 ftM is completely photoactivated after a 20-scc cxposurc to 254 n m light or five l-rainexposures to 366 n m light,as assessed by the disappearance of thc substrate, and the rate of its disappearance is independent of the temperature of the photolyzcd solution. Since photolysis of 4'-azidowarfafin at 254 n m is more efficient,studies with purified cytochromcs P450 arc conducted at this wavelength. However, in microsomal preparations 366 n m light is more efficientat photoactivating 4'-azidowarfarin and is thus preferentially used in photoaffinity labeling of microsomal-bound cytochromes P450. T w o criteriamust bc satisfiedin selecting a scavenger for photoactivatcd 4'-azidowarfarin. The potential scavcngcr should interact with and
[5]
PHOTOAFFINITY
0.4
I
PROBE
I
OF CYTOCHROMES I
F
i
P450 i
61 I
c"
E
o.s
0
/
v
J
O_
o
-.-t.
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-5
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i-.
