THE JOURNAL OF BIOLOGICAL CHEMISTRY
`0 1984 by The American Society of Biological Chemists, Inc.
`Kinetic Isotope Effects on Cytochrome P-450-catalyzed Oxidation
`Reactions
`EVIDENCE FOR T HE IRREVERSIBLE FORMATION OF AN ACTIVATED OXYGEN INTERMEDIATE OF
`CYTOCHROME P-448*
`
`Vol. 259, No. 5, Issue of March 10, pp. 3005-3010, 1984
`Printed I E U. S. A.
`
`Nobuhiro HaradaS, Gerald T. Miwag, John S. Walsh, and Anthony Y. H. Lu
`From the Department of Animal Drug Metabolism, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065
`
`(Received for publication, August 17, 1983)
`
`Substitution of deuterium for hydrogen on the a-
`tion of the cytochrome by NADPH-cytochrome P-450 reduc-
`carbon of 7-ethoxycoumarin results in an intrinsic iso-
`tase, and the binding of molecular oxygen to the reduced P-
`tope effect of approximately 14 during the cytochrome
`450-substrate complex (1). These studies have added to the
`P-448-catalyzed 0-deethylation of this substrate (G.
`detailed understanding about steps
`occurring in
`the early
`T. Miwa, J. S. Walsh, and A. Y. H. Lu (1984) J. Biol.
`stages of the P-450 catalytic cycle. However, details about
`Chem. 259, 3000-3004). This dramatic decrease in
`steps contained in the latter half of the catalytic cycle, such
`the C-H bond cleavage rate does not, however, alter
`as the activation of molecular oxygen and the addition of
`the rate of 7-ethoxycoumarin disappearance or the
`
`oxygen to the substrate, are much less well understood.
`rates of NADPH and oxygen consumption indicating
`Since the C-H
`bond cleavage step must occur after oxygen
`that the catalytic turnover of this enzyme is unaffected.
`Moreover, hydrogen peroxide formation and the con-
`activation, the study of primary kinetic isotope effects may
`centrations of an oxycytochrome P-448 complex (X,,,
`make possible the elucidation of mechanistic features around
`= 440 nm) are also unchanged demonstrating that the
`this step. To this end, we have previously studied the kinetic
`steady state concentrations of various oxy intermedi-
`isotope effects for N-demethylation reactions (2-4) and in the
`ates of cytochrome P-448 are also unchanged.
`preceding article we reported the extension of these studies
`
`An inescapable conclusion from these data is that an
`to 0-deethylation reactions catalyzed by this enzyme system
`irreversible step exists between the formation of these
`(5). In that study, the substitution
`of deuterium for the
`intermediates and the oxidation of the substrate. These
`hydrogen undergoing oxidative cleavage was shown to reduce
`data are in agreement with the view that an irrevers-
`the bond cleavage rate by approximately 12- to 14-fold for P-
`ible cleavage of the dioxygen bond precedes substrate
`oxidation. Moreover, the oxidatively competent form
`450 isozymes purified from
`the liver of rats induced with
`either PB or MC.
`of the cytochrome must then be committed to substrate
`oxidation. The latter conclusion is substantiated from
`In this paper, the consequences of this rate perturbation
`high performance liquid chromatography studies
`are examined. The rate of formation of a previously unre-
`which demonstrate the formation of a second metabo-
`(Table I,
`ported metabolite, 6-hydroxy-7-ethoxycoumarin
`lite, 6-hydroxy-7-ethoxycoumarin, from the deuter-
`compound VI), is greater from the deuterated substrate than
`ated substrate arising from the metabolic
`switching
`
`from the non-deuterated substrate without affecting the over-
`away from the 0-ethyl group to the aromatic ring of
`
`all turnover of cytochrome or the steady state levels of various
`this substrate.
`intermediates in the catalytic cycle. These data provide the
`first evidence for the existence of an irreversible kinetic step
`preceding substrate oxidation.
`
`The oxidation of xenobiotics by the cytochrome P-450-
`containing monooxygenase system involves a number of steps
`and the formation of several intermediates (1). In order to
`understand the detailed mechanism of this system, each step
`in the catalytic cycle must be elucidated. This has prompted
`extensive studies which have characterized, in detail, the
`substrate binding to oxidized P-450,l the first electron reduc-
`
`* Portions of this work were presented at a meeting of the Feder-
`ation of American Societies for Experimental Biology, New Orleans,
`LA, April 15-23, 1982. The costs of publication of this article were
`defrayed in part by the payment of page charges. This article must
`therefore be hereby marked "aduertisement" in accordance with 18
`U.S.C. Section 1734 solely to indicate this fact.
`$ Present address, National Institute of Environmental Health
`Sciences, National Institutes of Health, Research Triangle Park, NC
`27709.
`3 To whom correspondence should be addressed.
`I The abbreviations used are: P-450, generic name for the family
`of cytochrome P-450 isozymes; 7-EC, 7-ethoxycoumarin; other struc-
`ture abbreviations are given in Table I; PB, phenobarbital; MC, 3-
`methylcholanthrene; PB P-450, major liver isozyme isolated from
`phenobarbital-induced rats; P-448, major liver isozyme of cytochrome
`P-450 isolated from 3-methylcholanthrene-induced rats; HPLC, high
`pressure liquid chromatography.
`
`EXPERIMENTAL PROCEDURES
`(Table I, compound I) was pur-
`Substrates-7-Ethoxycoumarin
`chased from Aldrich or prepared from 7-hydroxycoumarin, V, and
`ethyl iodide (Aldrich). All isotopically labeled compounds were syn-
`thesized as detailed by Walsh et al. (6). 7-[l,l-2H~]Eth~xycoumarin,
`11, was prepared from [l,l-*Hz]ethyl iodide (Aldrichj and 7-hydrox-
`ycoumarin; isotopic purity, >95%, mass spectrum: m/z (m') 192. 7-
`[l-'4C]Ethoxycoumarin, 111, was synthesized from [l-"C]ethyl iodide
`(New England Nuclear) and 7-hydroxycoumarin; radiochemical pu-
`IV, was prepared from
`rity, 99.3%. 7-[1-'4C-l,l-2H~]Ethoxycoumarin,
`7-hydroxycoumarin and [l-14C-l,l-2H2]ethyl tosylate, in turn pre-
`pared from [l-14C-l,l-2H~]ethanol obtained through LiA12H4 (Merckj
`reduction of [l-14C]acetic anhydride (New England Nuclear); radi-
`ochemical purity, 99.4%. An authentic sample of the 7-[l,l-*H2]
`ethoxy-6-hydroxycoumarin metabolite, VI, was prepared by alkaline
`potassium persulfate oxidation of 11.
`Materials-NADPH
`and horseradish peroxidase were purchased
`from Sigma and 3-(p-hydroxyphenyl)propionic acid was obtained
`from Aldrich. Dilauroyl phosphatidylcholine was purchased from
`Serdary Research Laboratories (Ontario, Canada) and hydrogen per-
`oxide was obtained from Mallinckrodt. All other biochemicals were
`purchased from Sigma.
`Enzymes-Liver microsomes were obtained from PB- and MC-
`
`3005
`
`This is an Open Access article under the CC BY license.
`
`Auspex Exhibit 2009
`Apotex v. Auspex
`IPR2021-01507
`Page 1
`
`

`

`3006
`
`Structure of substrates and products m
`
`Deuterium Isotope Effect and
`
`
`
`
`TABLE I
`
`\ / RZ R1
`
`Substrates
`I
`I1
`I11
`IV
`Products
`V
`VI
`VI1
`VI11
`
`H
`
`
`
`CHZCH,
`C'HZCH,
`"CHZCH,
`"CZH&H3
`
`H
`C2H&H,
`"CH,CH,
`"C'HXH,
`
`H
`H
`H
`H
`
`OH
`OH
`OH
`
`induced male Long-Evans rats (PB, 75 mg/kg intraperitoneally for 4
`days; MC, 25 mg/kg intraperitoneally for 4 days) and MC-induced
`male Goldon Syrian hamsters (25 mg/kg intraperitoneally for 4 days)
`by standard methods (7). Hepatic PB P-450 and MC P-448 were
`purified from rat liver microsomal preparations as described by West
`et al. (8).
`NADPH-cytochrome P-450 reductase was isolated from PB-in-
`duced Long-Evans rats as described by Yasukochi and Masters (9).
`One unit of reductase activity is defined as the amount of enzyme
`catalyzing the reduction of cytochrome c at an initial rate of 1 nmol/
`min at 26 "C under the condition of Phillips and Langdon (10).
`Cytochrome c peroxidase was purified from bakers' yeast according
`to the procedure of Yonetani and Ray (11).
`7-Ethoxycoumarin 0-Deethylation Assay-The 0-deethylation ac-
`tivity of 7-ethoxycoumarin was measured fluorimetrically by follow-
`ing the formation of 7-hydroxycoumarin according to the method of
`Ullrich and Weber (12) with
`some modifications (13). A typical
`incubation mixture consisted of 0.075 nmol of cytochrome P-448,250
`units of NADPH-cytochrome P-450 reductase,
`30 pg of dilauroyl
`phosphatidylcholine, 0.25 pmol of 7-ethoxycoumarin, and 100 ymol
`of potassium phosphate buffer (pH 7.5) in a final volume of 1 ml.
`The reaction was started by addition of NADPH (150 nmol), and the
`mixture was incubated for 5 to 10 min at 23 "C. The reaction was
`terminated by addition of 0.15 ml of 15% trichloroacetic acid, and 7-
`hydroxycoumarin was extracted with 2 ml of chloroform. After vig-
`orous shaking for 10 min and centrifugation for 10 min, 1 ml of the
`chloroform layer was collected. 7-Hydroxycoumarin was recovered
`from the chloroform extract by addition of 3 ml of 10 mM NaOH
`containing 1 M NaCl. The 7-hydroxycoumarin was measured by the
`fluorescence intensity of this alkaline extract with an excitation of
`368 nm and an emission of 456 nm, and the concentrations were
`calculated from the standard
`curve prepared
`by adding known
`amounts of 7-hydroxycoumarin to the standard reaction mixture.
`7-Ethoxycoumarin Metabolite Profile-When
`the total metabolite
`profile of 7-ethoxycoumarin was examined, the chloroform extract
`from the reaction mixture was concentrated under a stream of nitro-
`gen gas and chromatographed by HPLC on a Zorbax ODS (Dupont)
`analytical column using an 8-min linear gradient from 40% methanol/
`water (v/v) to 100% methanol (14). Both substrate and metabolites
`were monitored by UV absorption a t 330 nm.
`Assays for Hydrogen Peroxide-Hydrogen peroxide formation was
`determined by three different methods. In most assays, the fluores-
`cence method of Zaitsu and Ohkuma (15) was used. As hydrogen
`peroxide in the terminated reaction mixture was not extracted by
`chloroform, 0.5 ml of the aqueous layer in the chloroform extraction
`was added to a mixture
`of 0.2 ml of 7.5
`mM 3-(p-hydroxy-
`pheny1)propionic acid, 2 ml of 0.15 M Tris-HC1 buffer (pH 8.51, and
`0.1 ml of 2 units/ml of horseradish peroxidase. After 10 min, the
`fluorescence emission intensity was measured at 404 nm with an
`excitation at 320 nm. The recovery of hydrogen peroxide was deter-
`mined by addition of a known amount of hydrogen peroxide to the
`reaction mixture. Hydrogen peroxide formation was also measured
`by cytochrome c peroxidase which forms a stable complex stoichio-
`metrically with hydrogen peroxide and by an oxidase meter (Yellow
`Springs Instrument Co. model 25) equipped with hydrogen peroxide
`
`Metabolic Switching
`
`electrode. The formation of cytochrome c peroxidase-hydrogen per-
`oxide complex was calculated from the hydrogen peroxide difference
`spectrum (A324-314nm) according to Yonetani (16) using an extinction
`coefficient of 42 mM" cm".
`Both methods gave the same results as
`that obtained by the fluorescence method, confirming the reliability
`of these three methods.
`Oxygen Consumption and NADPH Oxidase Assays-Oxygen con-
`sumption was determined by using an oxygraph (Gilson model 5-6H)
`equipped with a Clark oxygen electrode. NADPH oxidation was
`determined by following the decrease of absorbance at 338 nm instead
`of 340 nm because 7-ethoxycoumarin and 7-hydroxycoumarin have
`the same molar extinction coefficient a t 338 nm while NADPH shows
`the same absorbance (E338nm = 6.26 mM" cm")
`at both wavelengths
`(17).
`Steady State Levels of the Oxycytochrome P-450 Complex-The
`steady state levels of the oxy-P-450 complex in microsomes during 7-
`ethoxycoumarin metabolism were determined by the method of Wer-
`For this
`ringloer and Kawano (18) using
`= 42 mM-' cm".
`assay, various sources of microsomes (2.5 mg of protein) were added
`to the standard reaction mixture for the 7-ethoxycoumarin O-deeth-
`ylation assay except that 0.4 mM NADH was also included to reduce
`the cytochrome b,. The suspension was divided between two cuvettes
`and a base-line was recorded. NADPH (0.2 mM) was added to the
`sample cuvette, and the difference spectrum was recorded immedi-
`ately.
`content of cytochrome P-450 in microsomes
`Other Assays-The
`was determined according to Omura and Sat0 (19) using the absorp-
`tion coefficient of 91 mM" cm".
`Protein concentrations were deter-
`mined by the method of Lowry et al. (20) using bovine serum albumin
`as a standard.
`impact mass spectra of underivatized sam-
`Mass Spectra-Electron
`ples of the isotopically labeled ethoxycoumarin substrates, 7-[1,1-
`2Hz]etho~y-6-hydr~~y~~~marin
`metabolite and standard, VI, were
`obtained on a LKB model 9000 mass spectrometer. The operating
`conditions were: 70 eV ionization potential, 50 pA filament current,
`3.5 kV acceleration potential, and 250 "C source temperature. The
`samples were introduced into the source by way of the direct insertion
`probe.
`NMR Analysis-Proton NMR were obtained on a Varian 300-MHz
`superconducting spectrometer equipped with a Fourier transform
`accessory. Samples were dissolved in CDC13 and chemical shifts (parts
`per million) are presented relative to trimethylsilane.
`
`RESULTS
`Steady State Levels of the Oxy-P-450 Complex-Previous
`studies have established that deuterium substitution of the a-
`hydrogen that undergoes bond cleavage during the O-de-
`ethylation of 7-EC results in a deuterium isotope effect on
`V,,,, but not on K,, with various purified P-450 and microso-
`mal systems (5). Since deuterium substitution selectively re-
`duces the rate of the bond cleavage step, which occurs late in
`the catalytic cycle of the enzyme, an accumulation of inter-
`mediate P-450 complexes formed prior to this step would be
`expected during the oxidation of the deuterium-labeled sub-
`strate, 11. The steady state levels of one of these intermedi-
`ates, the oxy-P-450, can be easily measured by the method of
`Werringloer and Kawano (18). As shown in Table 11, the
`steady state levels of the oxy-P-450 complex in the metabo-
`lism of 7-EC as well as the observed deuterium isotope effects
`
`of the reaction are dependent on the sources of the microsomal
`preparations. Although the observed isotope effects in the 0-
`deethylation reaction were significant in all microsomal prep-
`arations, there are no differences in the steady state levels of
`oxy-P-450 complexes in the reaction with I or I1 demonstrat-
`ing the lack of further accumulation of the oxy-P-450 complex
`with the deuterated substrate. The steady state level of the
`oxy-P-450 complex does, however, change as a function of the
`pH of the reaction mixture (data not shown) as
`previously
`reported (18, 21).
`Stoichiometry Studies of 7-Ethoxycoumarin Metabolism
`the
`with the Cytochrome P-448-reconstituted System-Since
`slower rate of 0-deethylation following deuterium substitu-
`
`Auspex Exhibit 2009
`Apotex v. Auspex
`IPR2021-01507
`Page 2
`
`

`

`Deuterium Isotope Effect and Metabolic Switching
`TABLE I1
`Steady state levels of oxycytochrome P-450 complexes in the
`metabolism of isotopically labeled 7-ethoxycoumarin
`Various sources of microsomal preparations were used. Assay con-
`ditions are described under “Experimental Procedures.”
`Fraction of total P-450 as
`oxycytochrome P-450
`Substrate I Substrate I1
`%
`
`Isotope effect
`( U H / U D O )
`
`Microsomal
`preparation
`
`3007
`
`MC-treated rats
`11
`23
`
`
`
`PB-treated rats 3.85 23
`5.46
`45
`46
`MC-treated hamsters
`Isotope effects are expressed as relative rates at 0.25 mM substrate
`concentrations.
`
`10
`
`2.14
`
`
`
`Retentlon Time (min)
`
`Retention Time
`
`( m i n )
`
`+3.55
`+3.27
`
`TABLE 111
`Stoichiometry of 7-ethoxycoumarin metabolism with a cytochrome
`P-448-reconstituted system
`Assay conditions are described under “Experimental Procedures.”
`ANADPH
`A7-Hydroxycoumarin AH202
`AO,
`
`oxidation
`
`consumption formation
`formation
`nmol/5 min/reaction mixture
`-13.2
`+10.7
`-12.4
`+4.49
`
`Substrate
`-
`
`I
`I1
`
`-14.4
`-13.2
`
`u H / U D
`-
`
`1.09
`
`
`
`1.06 1.09
`
`
`
`2.38
`
`tion cannot be explained by the increase in the oxy-P-450
`level, stoichiometry experiments were carried out in order to
`determine the consequences of the primary isotope effect on
`the P-450 reaction
`cycle. A reconstituted monooxygenase
`system containing P-448 was used in all subsequent experi-
`ments. As shown in Table 111, deuterium-labeled 7-EC, 11,
`was oxidatively 0-deethylated to 7-hydroxycoumarin at a
`slower rate than the unlabeled substrate, I, giving rise to an
`observed primary deuterium isotope effect of 2.38. However,
`the formation of hydrogen peroxide, a product of uncoupling
`of the intermediate complexes of P-450 and O2 (22, 23) was
`virtually identical for both compounds. This result indicates
`that deuterium substitution does not induce the further ac-
`cumulation or the
`uncoupling of the P-45O-O2 complexes
`which are known to generate H202. Moreover, these results
`are consistent with those obtained on the steady state
`level
`of oxy-P-450 complexes in the metabolism of 7-EC. Table I11
`also shows that there are no differences in the utilization of
`NADPH and oxygen with the two substrates, indicating that
`deuterium substitution has not altered the
`overall oxidase
`activity of the system. These results suggest that an alternate
`metabolite of 7-ethoxycoumarin must be formed to explain
`the discrepancy in the material balance
`observed with the
`deuterated substrate.
`HPLC Analysis of 7-Ethoxycoumarin Metabolites-In order
`to determine the disposition of 7-EC during metabolism the
`total metabolite profile was examined by reverse phase HPLC
`14C-substrate, 111, was
`(Fig. 1). When the nondeuterated
`incubated with the purified P-448 system in the presence of
`profile monitored by UV absorption
`NADPH, the HPLC
`showed that 7-hydroxycoumarin was formed predominantly
`(retention time of 9.0 min) along with a trace amount of
`another metabolite (retention time of 9.7 min) (Fig. 1, upper
`left). The radiochromatogram (Fig. 1, lower left) demonstrated
`that the radiolabel was retained in the metabolite that was
`formed in trace quantity. On the other hand, when the deu-
`terated 14C-substrate, IV, was incubated the formation of 7-
`hydroxycoumarin was decreased
`approximately 2-fold con-
`sistent with the isotope effect of 2.38 determined fluorimet-
`
`R s ( a * m Time (min )
`Retentton T ~ m ( m n )
`FIG. 1. HPLC profile of the metabolites of isotopically la-
`beled 7-EC substrates. The deuterated (d2) or nondeuterated (do)
`7-[l-14C]ethoxycoumarin (0.5 mM) were incubated with a reconsti-
`tuted cytochrome P-448 system consisting of 0.3 nmol of cytochrome
`P-448, 1000 units of reductase, and 30 fig of dilauroyl phosphatidyl-
`choline in the presence of 3 mM NADPH at 37 “C for 5 min. The
`metabolites were extracted into chloroform and were concentrated
`under a stream of dry nitrogen. The concentrated metabolites were
`analyzed by HPLC under the conditions described under “Experi-
`mental Procedures.” The UV absorbance (upper portions) was mon-
`itored at 330 nm. Fractions were collected every 12 s and the radio-
`activity quantitated by liquid scintillation (lower portions). The re-
`tention times are approximately 9.0 min for 7-hydroxycoumarin, v,
`9.7 min for 6-hydroxy-7-ethoxycoumarin, VI1 or VIII, and 12.5 min
`for 7-ethoxycoumarin, 111 or IV.
`
`rically (Table 111). Concomitantly, a greater amount of the
`minor metabolite was formed with a retention time of 9.7 min
`(Fig. 1, upper right). The greater quantity
`of the minor
`metabolite could also be confirmed from the radiochromato-
`gram (Fig. 1, lower right).
`Identification of a Second Metabolite-The
`second metabo-
`lite was purified from an incubation mixture containing I1 by
`repetitive HPLC under the conditions described under “Ex-
`perimental Procedures.” The electron impact mass spectrum
`(Fig. 2, right) revealed that this metabolite had a molecular
`ion 16 amu larger than the substrate, I1 (left). Moreover, this
`16 amu difference was preserved in the two major fragment
`ions occurring at mlz 150 and 178 suggesting that hydroxyl-
`ation had occurred on the aromatic ring. The fragmentation
`of the metabolite is analogous to that observed for the sub-
`strate and is due to the sequential loss of ethylene and CO as
`depicted in Fig. 2.
`The 300-MHz NMR spectrum of the aromatic proton re-
`gion of this metabolite is shown in the lower portion of Fig. 3.
`
`Auspex Exhibit 2009
`Apotex v. Auspex
`IPR2021-01507
`Page 3
`
`

`

`3008
`
`100 4
`
`50
`
`30
`20
`10
`
`150 m/z 200
`
`100
`
`1
`
`L
`
`Deuterium Isotope Effect and Metabolic Switching
`A synthetic standard of the metabolite, VI, was prepared
`and analyzed by MS and NMR. Both spectra were identical
`with those of the metabolite (data not shown), confirming the
`structural assignment (VI).
`Stoichiometry of Substrate Utilization and Metabolite For-
`nation during the Metabolism of 7-Ethoxycoumarin-A radio-
`metric assay using I4C-labeled substrates was developed in
`order to quantitate the disappearance of substrate and $he
`formation of products. The substrate and the products were
`separated by reversed phase HPLC as
`shown in Fig. 1.
`7-Hydroxycoumarin, 6-hydroxy-7-ethoxycoumarin, and 7-
`ethoxycoumarin were eluted from the HPLC with retention
`times of 9.0,9.7, and 12.5 min, respectively. The consumption
`
`of 7-ethoxycoumarin and the formation of 6-hydroxy-7-ethox-
`ycoumarin were determined from the
`radioactivities of the
`corresponding HPLC peaks
`(Fig. 1, lower portion). Other
`experiments demonstrated that these
`two compounds were
`quantitatively extracted into CHCls and quantitatively re-
`covered during HPLC. The 7-hydroxycoumarin was measured
`fluorimetrically. Since 7-hydroxycoumarin has no radioactiv-
`ity due to the removal of 14C-labeled 0-ethyl group from the
`substrate, each determination can be carried out independ-
`As shown in Table IV,
`ently without mutual interference.
`there are little or no differences in the consumption of I11
`and IV demonstrating that deuterium substitution has not
`affected the overall turnover rate for 7-ethoxycoumarin me-
`tabolism. However, the 7-hydroxycoumarin metabolite was
`observed to form 1.9 times more slowly from IV by this assay,
`consistent with results obtained fluorimetrically (Table 111,
`substrate 11).
`In the metabolism of I11 by the P-448-reconstituted system,
`7-hydroxycoumarin is the major product (about 94% of total
`metabolites) while 6-hydroxy-7-ethoxycoumarin represents
`only about 5% (Table IV). In contrast, during the metabolism
`of IV, the formation of 7-hydroxycoumarin is decreased about
`2-fold compared to that observed for I11 while the formation
`of 6-hydroxy-7-ethoxycoumarin is increased about 5-fold rel-
`ative to that obtained from 111.
`
`60 40 70 30
`
`20
`IO
`0
`I 0 0
`
`~
`
`~
`
`~
`
`150
`
`m/z
`
`200
`
`d,-7EC Metabolite
`d y 7 E C
`FIG. 2. Mass spectra of deuterated 7-ethoxycoumarin (dz-
`7EC) and its metabolite. The deuterated 7-EC was incubated with
`the cytochrome P-448-reconstituted system as described under "EX-
`perimental Procedures." The new metabolite was separated from the
`chloroform-extracted metabolites by HPLC. Fractions containing the
`metabolite were pooled and electron impact MS were obtained from
`
`a 0.5-pg sample of the metabolite (right) and deuterated 7-EC (left). li
`
`DISCUSSION
`Stoichiometry studies of the metabolism of deuterated and
`nondeuterated 7-EC provide important information about the
`P-448 reaction cycle. Since the C-H
`bond cleavage step
`occurs late in the catalytic
`cycle of this enzyme (Fig. 4,
`conversion of VI to I), an increased steady state concentration
`of various intermediate cytochrome complexes (such as IV,
`V, and VI in Fig. 4) would be expected when the rate of this
`bond cleavage is decreased as a consequence of deuterium
`substitution.
`This perturbation did not, however, change the steady state
`levels of an oxy-P-448 intermediate (IV in Fig. 4) nor did it
`
`r
`
`
`
`TABLE IV
`Stoichiometry of substrate utilization and product formation in the
`metabolism of 7-ethoxycoumarin with cytochrome
`P-448-reconstituted system
`Deuterated and nondeuterated 7-[l-'4C]ethoxycoumarin were used
`as substrates. Substrates and products were separated by HPLC, and
`concentrations were determined by radioactivity and fluorescence.
`The assay conditions are described under "Experimental Procedures."
`
`
`
`A7-EC A7-Hydroxycoumar- A6-Hydroxy-7- Material
`EC formation balance
`Substrate consumption
`in formation
`
`
`nmol/5 minlreaction mixture
`+59.1
`+31.1
`
`+2.90
`+14.2
`
`%
`98
`80
`
`I11
`IV
`
`-63.1
`-56.3
`
`U H I U D
`
`1.12
`
`1.90
`
`0.20
`
`1
`
`l
`7 5
`
`,
`
`,
`
`,
`
`,
`
`l
`7 0
`
`,
`
`,
`
`c
`
`~
`6 5
`
`l
`
`#
`
`~
`
`8
`
`€
`
`PPM
`
`FIG. 3. 300-MHz nuclear magnetic resonance spectra of
`deuterated 7-ethoxycoumarin (d2-7EC) and its metabolite.
`The new metabolite was collected as described in the legend of Fig.
`1. NMR spectra were obtained with approximately IO-pg quantities
`of the metabolite (lower) and deuterated substrate (upper) dissolved
`in deuteroacetone.
`
`Of primary significance is the simplicity of the metabolite
`spectrum compared with the substrate (Fig. 3, upperportion).
`The doublets observed for C5 and Cs protons in the substrate
`have degenerated into singlets in the metabolite. Moreover,
`the C6 proton doublet of doublets observed in the substrate is
`absent in the metabolite. These data unequivocally demon-
`strate that hydroxylation has occurred at c6 on the aromatic
`ring.
`
`Auspex Exhibit 2009
`Apotex v. Auspex
`IPR2021-01507
`Page 4
`
`

`

`3009
`
`Deuterium Isotope Effect and Metabolic Switching
`process is nonproductive, however, only in the sense that the
`cause an increase in H202 production which would have been
`dissociation did not lead to an isotopically sensitive step.
`produced from higher steady state levels of intermediate com-
`( k ~ / k ~ = 13.5) for the O-de-
`The intrinsic isotope effect
`plexes IV or V (Fig. 4). Thus, to the extent that the catalytic
`ethylation of 7-EC by P-448 has been estimated from the
`cycle has been perturbed by deuterium substitution, there is
`observed deuterium and tritium isotope effect measurements
`no evidence for the reversibility necessary for the accumula-
`(5). We had previously suggested that a relatively high “com-
`tion of various oxy-P-448 complexes. These data are compat-
`mitment to catalysis” was responsible for the 7-fold suppres-
`ible with the irreversible bond cleavage of molecular oxygen
`isotope effect (observed DV/K = 2.0)
`sion of the intrinsic
`during the formation of an active oxygen-P-448 species such
`when 7-hydroxycoumarin was measured. The ratio of 7-hy-
`as the oxenoid intermediate, VI, depicted in Fig. 4.
`droxycoumarin to 6-hydroxy-7-ethoxycoumarin formed from
`The irreversible formation of this oxenoid intermediate
`I11 is 20 (Table IV). This is approximately the value estimated
`appears to commit this enzyme to the oxidation of the 7-EC
`earlier (5) for C, ( kH/k2 = 13) and demonstrates that kz chiefly
`
`substrate since deuterium substitution failed to slow the turn-
`reflects the dissociation-reassociation of 7-EC from the cata-
`over of the enzyme as assessed by 7-EC utilization, NADPH
`lytic site required for the formation of the 6-hydroxy-7-ethox-
`oxidation, or O2 consumption (Tables I11 and IV). The in-
`ycoumarin metabolite.
`creased formation of a second metabolite, 6-hydroxy-7-ethox-
`This phenomena of metabolic switching in the metabolism
`ycoumarin, which is normally formed in trace quantities rel-
`of deuterium-substituted compounds was first reported by
`ative to the 7-hydroxycoumarin metabolite (Table IV) dem-
`Horning et al. (25, 26) and Jarman et al. (27). They observed
`onstrates the strong commitment to catalysis of this active
`that deuterium substitution gave rise to different ratios in the
`oxygen intermediate.
`that a “V/K isotope effect
`metabolites formed from caffeine,
`antipyrine (25, 26), and
`It has been previously shown
`cyclophosphamides (27), and suggested the possibility that
`must be a consequence of a rapid interchange between the
`deuterium substitution on compounds metabolized by multi-
`deuterium labeled and unlabeled substrates on the
`enzyme
`ple alternative pathways rechanneled metabolism to pathways
`presence of an observed DV/K
`catalytic site (2). Thus, the
`away from the deuterated portion of the substrate. On the
`isotope effect for 7-EC along with the evidence for the irre-
`other hand, Lindsay Smith and Sleath (28)
`have recently
`versible formation of the oxene intermediate provide compel-
`demonstrated that several chemical model systems of P-450,
`ling arguments for the reversible binding of 7-EC to this
`including tetraphenylporphinatoiron (111) chloride and iodo-
`intermediate during catalysis. Consequently, these data indi-
`sobenzene, do not give metabolic switching even though the
`at least two forms of the
`cate that this substrate binds to
`large isotope effect observed for the 0-demethylation of tri-
`cytochrome (Fig. 4, I1 and VI) during its catalytic cycle.
`deuteriomethylanisole (kH/kD = 9.0) is similar in magnitude
`Northrop has developed equations that permit interpreta-
`to those reported here and by Jarman et al. (27). In addition,
`tion of isotope effect data on enzyme-catalyzed reactions (24).
`Bjorkhem (29) did
`not observe metabolic switching in
`the
`Equation 1 describes the relationship between the observed
`deuterium isotope effect, DV/K, and the intrinsic
`metabolism of deuterated lauric acid by P-450 in spite of a
`isotope
`significant deuterium isotope effect ( kH/kD = 3 to 4). Thus, it
`effect, k ~ / k D .
`is not yet clear whether this metabolic switching is a general
`phenomena and further stoichiometry studies would be nec-
`essary with other substrates and other
`isozymes of P-450
`before a generalization about the characteristics of the active
`oxygen form of cytochrome P-450 can be made.
`In conclusion, the commitment to catalysis term, C,, has
`to reflect the partitioning of the substrate, I,
`been shown
`between the 0-deethylated product, V, and the ring hydrox-
`ylated product, VII. In addition, the actual rate
`reduction
`caused by deuterium ( k ~ / k D = 13.5) could substantially be
`accounted for by a “metabolic switching”
`to a second, ring-
`hydroxylated metabolite (Table IV). This “metabolic switch-
`ing” occurs without consequence to the steady state levels of
`various P-450 intermediates and the overall turnover of the
`enzyme. We have interpreted these data to indicate that an
`irreversible oxygen-oxygen bond cleavage step occurs prior to
`substrate oxidation.
`
`The partition ratio, C,, is referred to as the “commitment to
`catalysis” by Northrop since C, is equal to the ratio
`kH/kz
`where kH is the rate constant for the C-H
`bond cleavage step
`leading to the 7-hydroxycoumarin metabolite and kz is the
`rate constant for the nonproductive dissociation of the P-450-
`ethoxycoumarin complex. The metabolic switching observed
`in the present study is simply another manifestation of this
`nonproductive dissociation of the P-448-7-EC complex. This
`
`RH
`
`d-
`FIG. 4. Proposed scheme for the cytochrome P-450 cata-
`lytic cycle. RH and ROH represents the 7-EC substrate and the 7-
`hydroxycoumarin product, respectively. R’OH represents the 6-hy-
`droxy-7-ethoxycoumarin metabolite. I, oxidized P-450; 11, substrate-
`bound oxidized P-450; 111, substrate-bound reduced P-450; IV, oxy-
`P-450 complex; V, 2-electron reduced oxy-P-450; VI, active oxygen-
`P-450 (oxene).
`
`Acknowledgments-We are indebted to Dr. Byron Arison for NMR
`spectra and Dr. William VandenHeuvel for obtaining the mass spec-
`tra. We wish to also gratefully acknowledge Lois Argenbright and
`Regina Wang for the purifie