Downloaded from
`
`http://www.jbc.org/
`
` by guest on May 16, 2016
`
`THE JOURNAL OF BIOLOGICAl, CHEMlln'RY
`Vol. 258, No. 7, Issue of April!O, pp. 4202-4207, 1983
`Printed i1t U.S.A.
`
`The Cytochrome P-450 Active Site
`REGIOSPECIFICITY OF PROSTHETIC HEME ALKYLATION BY OLEFINS AND ACETYLENES*
`
`Kent L. Kunze, Bonnie L. K. Mangold, Conrad Wheeler, Hal S. Beilan, and
`Paul R. Ortiz de Montellano*
`From the Department of Pharmaceutical Chemistry, School of Pharmacy, and Liver Center, University of California,
`San Francisco, California 94143
`
`(Received for publication, October 7, 1982)
`
`Hepatic microsomal cytochrome P~450 from phen~
`barbital~pretreated rats is inactivated during the me(cid:173)
`tabolism of linear olefins (ethylene, propene, and oc(cid:173)
`tene) and acetylenes (acetylene, propyne, and octyne).
`As expected from previous work, the inactivation is
`due toN-alkylation of the prosthetic heme group by the
`substrate. TheN-alkyl group in each adduct is formally
`obtained by addition of a porphyrin nitrogen to the
`terminal carbon and of an oxygen atom (as a hydroxyl
`function) to the internal carbon of the 17'-bond. The
`oxygen is shown here by 180 studies to be catalytically
`introduced by the enzyme. The olefins exclusively al·
`kylate the nitrogen of pyrrole ring D, but the acetylenes
`alkylate that of pyrrole ring A. Acetylene is an excep(cid:173)
`tion in that it reacts with more than one nitrogen.
`Circular dichroism studies of the ethylene adduct and
`of the ring D regioisomer of N-ethylprotoporphyrin IX
`obtained by alkylation of the prosthetic heme of he(cid:173)
`moglobin have been used to determine which face of
`cytochrome P-450 heme is alkylated by the unsaturated
`substrates. These results implicate an active site that
`is sterically encumbered in the region over pyrrole ring
`B and has a lipophilic binding site that accommodates
`chains of at least six carbon atoms over pyrrole ring C.
`
`The mechanism by which cytochrome P-450 transfers ac(cid:173)
`tivated oxygen to its substrates and the features of the active
`site that govern substrate binding have proven to be experi(cid:173)
`mentally elusive. The numerous studies of substrate selectiv(cid:173)
`ity available in the literature provide important information
`on the substrate preference of various isozymes but are not
`amenable to coherent interpretation in terms of the active site
`topology of any one isozyme. A salient exception is the recent
`exploration by Jerina and his collaborators, using the stereo(cid:173)
`chemistry of epoxidation of a large number of polycyclic
`aromatic hydrocarbons as the experimental probe, of the
`topology of cytochrome P-450c from rat liver (1, 2). Imai has
`
`*This work was supported by National Institutes of Health Grant
`GM-25515. Mass spectra were obtained at the Biomedical and Envi(cid:173)
`ronmental Mass Spectrometry Resource (Berkeley, CA) supported
`by National Institutes of Health Grant RR 00719 and P50 AM-19520.
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby
`marked "advertisement" in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`:J: Research Fellow of the Alfred P. Sloan Foundation (1978-1982).
`To whom correspondence should be addressed.
`
`independently investigated the topology of the analogous
`isozyme from rabbit liver (P-4481) by measuring the effect of
`one ligand on the binding of a second (3). Indirect and some(cid:173)
`tinles cumbersome methods such as these, in the absence of
`crystallographic structures for the cytochrome P-450 iso(cid:173)
`zymes, are the only means now available for the acquisition of
`topological information.
`The use of heme1 alkylation to probe the topology and
`mechanism of cytochrome P-450 is suggested by our finding
`that the prosthetic heme of the phenobarbital-inducible rat
`enzyme is alkylated during catalytic turnover of terminal
`olefms and acetylenes (4-7). Heme alkylation by olefms and
`acetylenes involves addition of an oxygen atom to one carbon
`of the 'IT-bond and of a heme pyrrole nitrogen to the other.
`TheN-alkyl group in the resulting adducts, which are isolated
`as the iron-free N-alkylprotoporphyrin IX derivatives, is a 2-
`hydroxyethyl function in the case of ethylene (4) and a 2-
`oxopropyl moiety in the case of propyne (5). The advantages
`of prosthetic heme alkylation as a probe of the enzyme are
`that (a) the heme, as an integral component of the active site,
`by definition does not perturb the normal catalytic sequence,
`(b) the heme provides an absolute set of coordinates within
`the active site to which topological data can be related, and
`(c) the heme, because of its intimate involvement with the
`catalytic process, provides "real time" information on the
`fleeting events occurring during catalysis. The use of pros(cid:173)
`thetic heme in this context requires that heme alkylation
`occur during, rather than subsequent to, the catalytic event.
`The alkylation reaction that accompanies the metabolism of
`terminal olefins and acetylenes, according to various lines of
`evidence, fulfills this condition (see introduction to the accom(cid:173)
`panying article) (8). Prosthetic heme alkylation has recently
`provided evidence in support of a nonconcerted, probably free
`radical, mechanism for olefin epoxidation (6, 8). The use of
`heme alkylation to investigate the orientation of substrates in
`the active site, suggested by our fmding that propyne reacts
`almost exclusively with the nitrogen of pyrrole ring A (5) but
`ethylene with that of either pyrrole ring C or D, is reported
`here (4). We have determined (a) the origin of the oxygen
`atom incorporated into the porphyrin adducts, (b) the abso(cid:173)
`lute stereochemistry of the adduct obtained with ethylene,
`and (c) the regiochemistry of heme alkylation by three olefms
`and three acetylenes. The results confirm that the enzyme
`catalytically initiates alkylation of its own prosthetic heme,
`establish which face of heme reacts with the substrates, and
`define key elements of the active site topology.
`
`1 The trivial name and abbreviation used are: heme, iron protopor(cid:173)
`phyrin IX regardless of the iron oxidation state; DDEP, 3,5-
`bis(carbethoxy)-2,6-dimethyl-4-ethyl-1,4-dihydropyridine.
`
`4202
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01564
`Page 1
`
`

`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on May 16, 2016
`
`Regiospecificity of Heme Alkylation
`2
`MATERIALS AND METHODS
`
`4203
`
`RESULTS
`Octene, octyne, and propene decrease the concentration of
`cytochrome P-450 when incubated with hepatic microsomes
`from phenobarbital-pretreated rats. The time-dependent de(cid:173)
`crease in spectroscopically measured cytochrome P-450 is
`essentially complete within 30 min. The total cytochrome P-
`450 content was reduced at this time by 22 ± 2, 32 ± 6 and 32
`± 1%, respectively. Enzyme loss is prevented by omission of
`either NADPH or the substrate from the incubation mixture.
`Treatment of phenobarbital-induced rats with octene (500
`mg/kg), octyne (500 mg/kg), and propene (40% in air) resulted
`in the accumulation of abnormal liver porphyrins. The por(cid:173)
`phyrins were converted to their metal-free dimethyl esters by
`treatment with acidic methanol and were extracted and puri(cid:173)
`fied by thin layer and high pressure liquid chromatography
`according to previously developed protocols (4, 5). Each por(cid:173)
`phyrin migrated as a single band or peak during the multiple
`purification steps. The electronic absorption spectra of the
`porphyrins, both in the metal-free and zinc-complexed state,
`were characteristic of N-alkylprotoporphyrin derivatives (Amax
`free base: 418, 512, 546, 592, and 652 nm; zinc complex: 432,
`545, 592, and 630 nm). A long wavelength shoulder on the
`Soret band was only observed with the zinc complex of the
`porphyrin obtained with octyne.
`The molecular ion (MH+) in the field desorption mass
`spectrum of each of the metal-free porphyrins (propene ad(cid:173)
`duct, mje 649; octene adduct, m/e 719; octyne adduct, m/e
`717) corresponds, as found before with ethylene and propyne
`(4, 5), to the sum of the molecular weights of the dimethyl
`ester of protoporphyrin IX plus the destructive agent plus an
`oxygen atom. The source of the oxygen atom, however, has
`remained unknown. The ethylene adduct has therefore been
`generated in vitro under an atmosphere highly enriched in
`11'02. The resulting adduct was purified by the standard pro(cid:173)
`cedure and was compared by field desorption mass spectrom(cid:173)
`etry with the porphyrin previously obtained from ethylene(cid:173)
`treated rats (4). The mass spectra of the two samples were
`obtained on the same day and with the same emitter to
`minimize instrumental variations (Table I, Miniprint). The
`porphyrin obtained in vivo has the expected molecular ion at
`m/e 634 and monoprotonated molecular ion at mje 635, but
`the porphyrin obtained in the incubation with 180 2 exhibits a
`large monoprotonated molecular ion at m/e 637 with the
`attendant isotope peak at 638 and only a minor peak at m/e
`635. The ratio of protonated to unprotonated molecular ions
`for N-alkylporphyrins has been found to be highly variable
`(12). The two mass unit difference observed here nevertheless
`clearly establishes that approximately 85% of the porphyrin is
`labeled with 1802. The 15% of unlabeled oxygen incorporated
`into the adduct is not unexpected because the gentle purging
`required to maintain enzyme activity is not sufficient to re(cid:173)
`move all the dissolved oxygen from the incubation mixture.
`Alkylation of prosthetic heme by ethylene, acetylene, and
`propyne involves formal addition of oxygen to one end of the
`w-bond and of a heme pyrrole nitrogen to the other (4-6). This
`reaction pattern could give rise in the present instance to a
`
`2 Portions ofthis paper (including "Materials and Methods," Tables
`I and II, and Figs. 1-3) are presented in miniprint at the end of this
`paper. Miniprint is easily read with the aid of a standard magnifying
`glass. Full size photocopies are available from the Journal of Biolog(cid:173)
`ical Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request
`Document No. 82M-2726, cite authors, and include a check or money
`order for $3.20 per set of photocopies. Full size photocopies are also
`included in the microfilm edition of the Journal that is available from
`Waverly Press.
`
`total of eight distinct porphyrins (not counting stereoisomers)
`if each of the four nonidentical nitrogens reacts with the two
`ends of the asymmetric w-bond. The 360 MHz NMR spectra
`of the zinc-complexed octene, octyne, and propene adducts
`(Fig. 1, Miniprint), however, clearly establish that essentially
`only one isomer is actually formed in each instance. The
`formation at best of traces (approximately 5%) of other iso(cid:173)
`mers is indicated by the presence of essentially only one set of
`signals for each proton. The observed adducts result from
`addition of a nitrogen to the terminal carbon of the w-bond.
`The spectrum of the octyne adduct, for example, exhibits the
`2-proton singlet at -4.37 ppm expected for terminal N-alkyl(cid:173)
`ation rather than the one-proton multiplet expected for the
`internal alkylation product. Terminal N-alkylation is likewise
`confirmed for the octene and propene adducts by the presence
`of two-proton multiplets in the appropriate region of their
`NMR spectra (Fig. 1).
`The N-alkyl proton signals in each spectrum have been
`identified by spin decoupling experiments. Irradiation of the
`multiplet at -5 ppm in the ABM~ spin system of the N-(2-
`hydroxypropyl) moiety in the propene adduct (Fig. 1) has no
`effect on the doublet at -1.3, but causes subtle changes in the
`0.9 ppm region. Irradiation of this region reduces the methyl
`doublet at -1.3 ppm to a singlet and the 8-line methylene
`proton pattern at -5 ppm to 4 lines. The methylene protons
`therefore are nonequivalent and geminally coupled (Jgem = 15
`Hz). The different vicinal coupling constants of the two meth(cid:173)
`ylene protons with the methine proton (Jvic = 2 and 7Hz)
`indicate that rotation about the carbon-carbon bond is slow
`on the NMR time scale at room temperature. Similar results
`are obtained with the octene adduct (see Ref. 8). The remain(cid:173)
`ing alkyl group proton signals for the octene and octyne
`pigments are identified in Table II (see Miniprint). The pres(cid:173)
`ence of two chiral centers in the olefin adducts make each set
`of methylene protons, including those of the propionate side
`chains, diastereotopic. The C-3 and C-4 pairs of methylene
`protons of the N-alkyl group in the octene adduct are suffi(cid:173)
`ciently dissimilar to be resolved in the NMR spectrum.
`The porphyrins isolated here from rats treated with pro(cid:173)
`pene, octene, and octyne, like those previously obtained with
`ethylene (4) and propyne (5), result from highly regioselective
`alkylation of a single pyrrole nitrogen. A technique has been
`developed to assign such isomers (11) and has been used to
`identify the nitrogen alkylated by propyne (5). The method
`hinges on the fact that an N-alkyl group on a porphyrin
`pyrrole ring causes the signals associated with the peripheral
`substituents of that ring to appear at higher field in the NMR
`spectrum relative to their position when on a nonalkylated
`ring. This reflects the different relationship of the substituents
`relative to the porphyrin ring current brought about by the
`associated tilting of the alkylated ring. The two internal vinyl
`protons of the octyne adduct, each a 4-line signal due to spin
`coupling with the terminal vinyl protons, differ in chemical
`shift by 0.3 ppm, while the same protons in the octene,
`propene, and ethylene (4) adducts differ by only 0.06 ppm
`(Fig. 1). Conversely, the four methylene protons adjacent to
`the porphyrin ring (4.0-4.4 ppm) are chemical shift equivalent
`in the spectrum of the octyne adduct, as are the four meth(cid:173)
`ylene protons adjacent to the propionate carboxyl groups (3.0
`to 3.3 ppm), but these protons are widely separated in the
`spectra of the three olefin adducts. The octyne adduct there·
`fore bears theN-alkyl group on either ring A orB (the vinyl(cid:173)
`substituted rings), whereas the olefin adducts are alkylated on
`either ring CorD (the rings with propionic acid side chains).
`In order to differentiate between rings A and B or C and D
`it is necessary to specifically assign the six methyl and four
`meso proton signals in each spectrum (11). This is done by
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01564
`Page 2
`
`

`
`4204
`
`Regiospecificity of Heme Alkylation
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on May 16, 2016
`
`-(CHz),CH,
`-CH3
`-H
`R·
`Fw. 4. Porphyrins derived from prosthetic heme adducts.
`The porphyrins derived from the prosthetic heme adducts with eth(cid:173)
`ylene (la), propene (lb}, octene (lc), acetylene (2a), propyne (2b)
`(5), and octyne (2c). Except for acetylene, which reacts with more
`than one nitrogen, the nitrogen alkylated is that shown in the struc(cid:173)
`tures.
`
`450
`
`425
`.000
`'Mlvelenllfh ( nm)
`Fm. 5. Circular dichroism spectra. Circular dichroism spectra
`of (a) the zinc-complexed dimethyl ester of N-(2-hydroxyethyl)(cid:173)
`protoporphyrin IX from ethylene-treated rats and (b) the ring D
`isomer of dimethyl esterified chlorozinc N-ethylprotoporphyrin IX
`from DDEP-treated rats. The spectra were recorded in CH2Clz.
`
`475
`
`dimethyl ester of the ethylene adduct exhibits bands of the
`same sign and at the same positions as does the spectrum of
`the ring D isomer of chlorozinc N-ethylprotoporphyrin IX
`(dimethyl ester) (Fig. 5) obtained by alkylation of the pros(cid:173)
`thetic heme of hemoglobin with ethylhydrazine (14).3
`
`DISCUSSION
`Oxidation of terminal olefins by the reactive iron-coordi(cid:173)
`nated species of atomic oxygen produced by catalytic turnover
`of cytochrome P-450 results in epoxide formation and heme
`N-alkylation. Heme alkylation, like olefin epoxidation, re(cid:173)
`quires catalytic turnover of the enzyme in the presence of
`NADPH and oxygen (7), is inhibited by carbon monoxide and
`SKF-525A (7), and, as shown here (Table I), results in incor(cid:173)
`poration of 1 atom of molecular oxygen into theN-alkyl group
`of the heme adduct. The parallels between epoxidation and
`heme alkylation argue that both reactions spring from inter(cid:173)
`action of the olefin with a single (or very closely related)
`activated oxygen species. This mechanistic analogy underlies
`the use of heme alkylation as a probe of the mechanism by
`which cytochrome P-450 oxidizes w-bonds. Of particular top(cid:173)
`ological relevance is the fact that catalytic incorporation of
`molecular oxygen into the olefin-derived N-alkyl group con·
`strains the spatial relationship of the olefin relative to the
`prosthetic heme group during the reaction because the inter(cid:173)
`nal carbon of the unsaturated bond must be juxtaposed with
`
`3 Ortiz de Montellano, P. R., Kunze, K. L., and Beilan, H. S. (1983)
`J. Bioi. Chem. 258,45-47
`
`defining a unique set of structural connectivities based on the
`fact that (a) nuclear Overhauser enhancement of meso proton
`signals is observed when adjacent methyl, internal vinyl, or
`benzylic methylene protons are irradiated, and (b) they meso
`proton has a shorter T 1 than the other three meso protons,
`whereas the two methoxy group protons relax more slowly
`than the ring methyls. The identification of the nitrogen
`alkylated by ethylene, the most difficult of the examples in
`this study, is outlined below to illustrate the logic involved.
`The y meso proton at 10.322 ppm (Fig. 2, Miniprint) in the
`NMR spectrum of the ethylene adduct (the full spectrum has
`been published) (4) is identified by its short T 1 (Table II,
`Miniprint) and by the enhancement of its intensity upon
`irradiation of the benzylic methylene protons at 4.3 ppm. One
`ester methyl (3. 705 ppm) is identified by its long T, (Table II)
`and the other (3.555 ppm), which overlaps with a ring methyl
`signal, by integration. Further assignments are hampered by
`the fact that two of the remaining meso proton signals are
`superimposed (Fig. 2). Irradiation of three separate methyls
`enhances the signal due to the overlapping meso protons. One
`of the two protons in question must therefore be at the 8 meso
`position. Sequential spin decoupling of each set of methyl
`group protons to determine which ones are spin-coupled (J
`= 0.8Hz) to internal vinyl protons identifies the 1 and 3
`methyls. A distinct sharpening of the 4-line signal due to an
`internal vinyl proton is observed when these two methyls are
`decoupled (middle two spectra, Fig. 3). Because a nuclear
`Overhauser enhancement of the overlapping meso proton
`signal was observed when the now identified 1- and 3-methyl
`protons were irradiated, the overlapping protons must be
`those at the a and 8 meso positions. The remaining meso
`proton (10.236 ppm), by difference, is that at the P position.
`This is confmned by enhancement of its signal when the
`internal vinyl protons are irradiated. Since the p meso proton
`is flanked by the 5-methyl group, the dipolar coupling ob(cid:173)
`served in the nuclear Overhauser experiment between the two
`assigns the latter (3.555 ppm). The 8-methyl group (3.388
`ppm) can then be identified because it is the only methyl
`group not yet assigned. Irradiation at 8.17 ppm (differential
`irradiation of the two internal vinyl protons was not possible)
`more strongly enhanced the f3 than the a meso proton signal.
`Irradiation at 8.22 ppm, on the other hand, more strongly
`enhanced the a signal. The vinyl proton dipolar coupled to
`the f3 meso proton is therefore the one ·centered at 8.17 ppm
`(that on the 4-vinyl). The 3-methyl group on the same ring as
`the 4-vinyl is then located by spin decoupling. The final
`complete meso and methyl assignments are given in Table II.
`The porphyrins obtained with octyne, octene, and propene
`were similarly analyzed. The results of these experiments are
`also given in Table II. It was not necessary to examine the
`coupling of the internal vinyl and meso protons in the propene
`and octene adducts, nor the existence of spin coupling between
`the internal vinyl and methyl protons in the octyne adduct, to
`make the assignments.
`A single methyl group signal is shifted upfield (Table II)
`relative to its expected position in each of the adducts. The
`range of chemical shifts for the 3- and 5-methyl signals are
`essentially invariant but the 1-methyl signal of the octyne
`adduct is approximately 0.2 ppm upfield from its position in
`the other three porphyrins. The octyne adduct therefore is
`alkylated on ring A (Fig. 4). The 8-methyl signals of the three
`olefin adducts likewise occur at significantly higher field (0.15
`ppm) than the 8-methyl signal of the octyne adduct (or of the
`three isomers of N-methylprotoporphyrin IX not alkylated on
`ring D) (11). The three olefin adducts therefore are alkylated
`on ring D (Fig. 4).
`The circular dichroism spectrum of the zinc-complexed
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01564
`Page 3
`
`

`
`Regiospecificity of Heme Alkylation
`
`4205
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on May 16, 2016
`
`the internal acetylenic carbon is anchored by reaction with
`the iron-bound oxygen, orientation of the terminal carbon
`toward the nitrogen of ring D forces the hydrocarbon tail of
`the substrate into the region over pyrrole ring B. If, on the
`other hand, the terminal carbon is placed over the nitrogen of
`ring A, the hydrocarbon chain extends over the region defined
`by ring C. The obse:cved acetylene reaction regiochemistry
`thus requires a lipophilic binding site able to accommodate
`chains of at least six carbons over pyrrole ring C. Independent
`evidence for such a binding region is provided by the fact that,
`if the internal carbon of the "IT-bond of an olefin is similarly
`fixed over the iron-bound oxygen and the terminal carbon
`over the nitrogen of ring D, the hybridization state of the
`internal carbon (sp2 going to sp3
`) places the hydrocarbon
`chain in the region over pyrrole ring C.
`In order to translate the regiochemical data into a specific
`active site geometry, it is necessary to know on which side of
`the prosthetic heme group oxygen activation and prosthetic
`heme alkylation occur. Heme is a prochiral molecule that
`gives rise to enantiomeric configurations when the two coor(cid:173)
`dination sites on the iron are differentially occupied. We have
`recently used the circular dichroism spectra of the ring C
`isomers of N-ethylprotoporphyrin IX obtained from reaction
`of cytochrome P-450 with DDEP (16) and of hemoglobin with
`ethylhydrazine (14) to establish that the prosthetic heroes in
`the two proteins have the same orientation relative to the
`fifth iron ligand. 3 Analogous comparison of the ethylene ad(cid:173)
`duct (alkylated on ring D) with the corresponding isomer of
`N-ethylprotoporphyrin IX (Fig. 5) shows that the olefin reacts
`with the same face of heme as DDEP. A model of the active
`site that incorporates the absolute geometry of the prosthetic
`heme, the presence of a steric constraint over pyrrole ring B,
`and the presence of a lipophilic channel over pyrrole ring C,
`is given in Fig. 6.
`The regiochemistry of prosthetic heme alkylation by one
`branched acetylene, 1-chloro-3-ethyl-1-penten-4-yn-3-ol (eth(cid:173)
`chlorvynol), is known (13). This sedative hypnotic reacts with
`at least three of the pyrrole nitrogens, including those of rings
`A and B. If the same cytochrome P-450 isozyme is inactivated
`by ethchlorvynol as by unbranched acetylenes, a fact that
`remains to be established, the active site structure must
`permit a much
`lower alkylation regiospecificity when
`"globular" rather than unbranched substrates are involved.
`The regiospecificity observed with propyne and octyne is
`difficult to reconcile, in fact, with metabolism of ethchlorvynol
`by the same hemoprotein unless the active site has some
`conformational flexibility. The constraints on reactions in one
`conformation are unlikely to be the same as those in another.
`A conformationally flexible active site would not be surprising
`for a catalytic system as complicated and promiscuous as
`cytochrome P-450 in view of the fact that even myoglobin, a
`dedicated monofunctional hemoprotein, apparently undergoes
`active site conformational breathing (17).
`The active site model proposed here predicts that heme
`alkylation should result from oxidation of the re face of the
`double bond in 1-octene, the exposed face when the internal
`carbon is fixed over the iron and the terminal carbon is over
`pyrrole ring D. The stereochemical studies reported in the
`accompanying paper confirm this prediction and furthermore
`establish that the opposite (si) face of the olefin can be
`oxidized but does not result in heme alkylation, a result
`consistent with the proposed active site structure (8).
`The model formulated for the active site leaves unexplained
`the fact that ethylene does not detectably alkylate the nitro(cid:173)
`gen of pyrrole ring C. The absence of ring C alkylation by
`olefins larger than ethylene is readily explained because this
`would involve intrusion of their substituents into the sterically
`
`the activated oxygen that initiates the reaction.
`The structures of the propene, octene, and octyne prosthetic
`heme adducts are formally obtained by addition of an oxygen
`(as a hydroxyl group) to the inside carbon of the "IT-bond and
`of a nitrogen from the protoporphyrin IX framework of heme
`to the terminal carbon (Fig. 4). These structures, in conjunc(cid:173)
`tion with those of the ethylene (4), acetylene (6), and propyne
`(5) adducts, clearly establish that reaction of the heme nitro(cid:173)
`gen with the terminal (unsubstituted) carbon of the "IT-bond is
`overwhelmingly favored. High regiospecificity is also observed
`with respect to the nitrogen of the heme that is alkylated. The
`three olefins (ethylene, propene, and octene) react almost
`exclusively with the nitrogen of pyrrole ring D but the two
`terminal acetylenes (propyne and octyne) react with that of
`pyrrole ring A (Fig. 4). Only acetylene, among the linear
`unsaturated hydrocarbons so far tested, is not highly regia(cid:173)
`specific and alkylates at least two of the nitrogens (6). The
`high specificity of the alkylation reaction points to a well
`defined active site topology.
`If one end of the "IT-bond (the substituted end if a substituent
`is present) is fixed approximately over the activated oxygen
`and the oxygen reacts with the "IT-bond but remains bound to
`the iron until heme alkylation occurs, additional active site
`stereoelectronic constraints must exist that prevent reaction
`of the ethylene with a nitrogen other than that of pyrrole ring
`D. This assumes, of course, that alkylation specificity is not
`due to an inherent electronic difference in the reactivity of
`the four nitrogens. Three observations indicate that even if
`such an intrinsic difference in nitrogen reactivity exists it is
`not a major determinant of regiochemical specificity: (a) N(cid:173)
`alkylation in a chemical model proceeds without regiospeci(cid:173)
`ficity (15), (b) N-alkylation of cytochrome P-450 heme by 4-
`alkyldihydropyridines (16) and ethchlorvynol (13) occurs with
`relatively low specificity, and (c) propyne and octyne, in
`contrast to ethylene, propene and octene, react almost exclu(cid:173)
`sively with the nitrogen of pyrrole ring A. The stereoelectronic
`constraints on substrates in the active site must allow acety(cid:173)
`lene a greater degree of freedom than ethylene in order to
`explain the lower regiospecificity of acetylene. This can be
`achieved if a protein residue located over pyrrole ring B blocks
`the approach of the laterally extended hydrogens of the rela(cid:173)
`tively bulky ethylene terminus to the nitrogen of ring A but
`does not interfere with that of the cylindrical, sterically com(cid:173)
`pact, acetylene functionality (Fig. 6).
`Sterle congestion over pyrrole ring B provides a ready
`explanation for the reaction of acetylene with multiple nitro(cid:173)
`gens (presumably including those of pyrrole rings A and D),
`whereas propyne and octyne only react with that of ring A. If
`
`FIG. 6. Model proposed for the active site of the phenobar(cid:173)
`bital-inducible cytochrome P-450 isozyme inactivated by un(cid:173)
`branched olefins and acetylenes. Oxygen activation and heme
`alkylation occur on the reader's side of the heme in the perspective
`given in the model. Definition of the absolute stereochemistry of the
`heme in the active site is intended.
`
`Boehringer Ex. 2016
`Mylan v. Boehringer Ingelheim
`IPR2016-01564
`Page 4
`
`

`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on May 16, 2016
`
`4206
`
`Regiospecificity of Heme Alkylation
`
`encumbered pyrrole ring A/B region, but the failure of eth(cid:173)
`ylene to react cannot be explained in the same manner. The
`model proposed here also does not explicitly incorporate re(cid:173)
`giochemical imperatives defmed by the still unknown detailed
`mechanism of the oxidation reaction. The regiochemical spec(cid:173)
`ificity, for example, may be affected if alkylation by acetylenes
`involves an iron-carbene intermediate or if that mediated by
`olefms proceeds through an intermediate in which the iron is
`simultaneously bound to the oxygen and to the terminal
`carbon ofthe original 'IT-bond (7). It is to be hoped that future
`refinements of the active site model, which is consistent with
`all of the information now available, will confirm its validity
`and improve its predictive value.
`
`REFERENCES
`
`1. Jerina, D. M., Michaud, D. P., Feldman, R. J., Armstrong, R. N.,
`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, Wiley(cid:173)
`Interscience, New York
`2. Yagi, H., and Jerina, D. M. (1982) J. Am. Chem. Soc. 104, 4026-
`4027
`3. Imai, Y. (1982) J. Biochem. (Tokyo) 92, 77-88
`4. Ortiz de Montellano, P.R., Beilan, H. S., Kunze, K. L., and Mico,
`B. A. (1981) J. Biol. Chem. 256, 4395-4399
`
`5. Ortiz de Montellano, P. R., and Kunze, K. L. (1981) Biochemistry
`20, 7266-7271
`6. Ortiz de Montellano, P. R., Kunze, K. L., Beilan, H. S., and
`Wheeler, C. (1982) Biochemistry 21, 1331-1339
`7. Ortiz de Montellano, P. R. (1983) in Bioactivation of Foreign
`Compounds (Anders, M. W., ed) Academic Press, New York, in
`press
`8. Ortiz de Montellano, P.R., Mangold, B. L. K., Wheeler, C., Kunze,
`K. L., and Reich, N. 0. (1983) J. Biol. Chem. 258, 4208-4213
`9. Ortiz de Montellano, P. R., Mico, B. A., Mathews, J. M., Kunze,
`K. L., Miwa, G. T., and Lu, A. Y. H. (1981) Arch. Biochem.
`Biophys. 210, 717-728
`10. Ortiz de Montellano, P. R., and Mico, B. A. (1980) Mol. Phar(cid:173)
`macal. 18, 128-135
`11. Kunze, K. L., and Ortiz de Montellano, P.R. (1981) J. Am. Chem.
`Soc. 103, 4225-4230
`12. Ortiz de Montellano, P.R., and Kunze, K. L. (1980) J. Biol. Chem.
`255, 5578-5585
`13. Ortiz de Montellano, P.R., Beilan, H. S., and Mathews, J. M.
`(1982) J. Med. Chem. 25, 1174-1179
`14. Augusto, 0., Kunze, K. L., and Ortiz de Montellano, P. R. (1982)
`J. Bioi. Chem. 257, 6231-6241
`15. Ortiz de Montellano, P.R., and Kunze, K. L. (1981) J. Am. Chem.
`Soc. 103, 6534-6536
`16. Ortiz de Montellano, P. R., Beilan, H. S., and Kunze, K. L. (1981)
`Proc. Natl. Acad. Sci. U. S. A. 78, 1490-1494
`17. Caughey, W. 8., Shimada, H., Miles, G. C., and Tucker, M.P.
`(1981) Proc. Natl. Acad. Sci. U.S. A. 78, 2903-2907
`
`Kent L. J(unz~, Bonnie L~ X~ Manqold., Conra:d Wbe~ler .. kl s.
`Beilan, and Paul R~ Ortis de Montellano.
`
`~~~
`'l'he followin9 che•icala were uaed without further purification
`except for oct.ene, which was dlatilled prior to \Ute: octene tAldrieh
`;~~~i.~~· ~~-~!:-"0: ci~:!:h~:. ~!vil~on(s=~~~!r c~::t~:}!, J~~=d:nd
`••thanol and hexane (Burdick and Jicuon) l RADPll, lfAnP- glueose-6-
`phoephate, and qlucose-6-phoepha:te dehydrogenase (S~9aa Che•. Co. •.
`Tetrahydrofutan vas diGUlled froa sodiu• under an ataosphtH'e of
`nitroqen iMIMtdiately pr io£ to use.
`been ;:::~hot1~o~-~ 7:~U~i!!0:f~emi;·11 t~!:!!:~ :~:t::l:h:ave
`apeettoaoopie meaa.urntent: ot t:ime and NADPB dependent loas of eyto(cid:173)
`chro•e P-450 {9). Miero.OMa ftoa aodiu., phenobacbital prett•ated (80
`Jlg/l:g) 250 9 Hle Spraque-DAwley rata were used in t~ae expe-riJHnts.
`tncubatlon •lxturea contained llicrosoaal