Toxicology 118 (1997) 93-113

Molecular modelling of CYP2El enzymes from rat, mouse and

man: An explanation for species differences in butadiene
metabolism and potential carcinogenicity, and rationalization of
CYP2E substrate specificity

David F.V. Lewisa,*, Michael G. Birdb, Dennis V. Parke”

“Molecular Toxicology Group, Centre for Toxicology, School of Biological Sciences, University of Surrey, Guildford,
Surrey GU2 5XH, UK
bToxicology Division, Exxon Biomedical Sciences, Inc., Mettlers Road, CN 2350, East Millstone, New Jersey 08875-2350, USA

Accepted 17 November 1996

Abstract

Molecular modelling of substrates of cytochrome P4502El (CYP2El) within the putative active site region of
CYP2El constructed from the CYP102 crystal structure is reported. Structural characteristics of CYP2El substrates,
such as molecular size, energy levels and polarity, calculated via molecular orbital procedures provide correlations
with toxicity and carcinogenicity; and species differences in CYP2El-mediated metabolism are rationalized in terms
of interactions with putative active site amino acid residues, including Thr-437 and Phe-181. In particular, the
activation of buta-1,3-diene can be explained by active site modelling with CYP2El enzymes sequenced from rat,
mouse and man, where there is a non-conservative change T437H between rodent and human isozymes, together with
a conservative change 1438V between mouse and rat CYP2El. 0 1997 Elsevier Science Ireland Ltd.

Keywords: CYP2EI enzymes; Butadiene metabolism; Carcinogenicity

1. Introduction

Abbreviations: CYP, cytochromes P450; AE, energy of 1.1. CYP2E enzymes

LUMO - energy of HOMO; EH, epoxide hydrolase; HOMO,
highest occupied molecular orbital; LUMO, lowest unoccupied
The cytochromes P45O(CYP) constitute a su-
molecular orbital; MEP, molecular electrostatic potential;
perfamily of over 400 heme-thiolate enzymes
QSAR, quantitative structure-activity relationship; ROS, reac-
tive oxygen species. which have been shown to be present in almost all
* Corresponding author. forms of life, and are involved primarily in the

0300-483X/97/$17.00 0 1997 Elsevier Science Ireland Ltd. All rights reserved.

PII SO300-483X(96)03583-4
94 D.F. V. Lewis et al. / Toxicology 118 (1997) 93- 113

Table 1
Characteristics of CYPZE substrates

Chemical AE (eV) iu (D) d (A) Comments

1. Paracetamol 8.71 4.513 6.4 (6.43) Weak carcinogen (hepatotoxic)

2. p-Nitrophenol 9.01 5.262 6.0 (6.00) Non-carcinogen
3. Aniline 9.16 1.541 5.6 (5.65) Weak carcinogen
4. p-Xylene 9.55 0.070 6.0 (6.08) Carcinogen
5. Dimethylsulfoxide 9.60 4.488 5.2 (5.13) Weak carcinogen
6. Isoniazid 9.16 1.563 6.1 (6.16) Carcinogen
7. Pyridine 10.07 1.975 5.2 (5.28) Weak carcinogen
8. Azoxymethane 10.09 5.085 5.1 (5.18) Weak carcinogen
9. Imidazole 10.14 3.601 4.9 (4.93) Non-carcinogen
10. Benzene 10.20 0.001 5.4 (5.36) Carcinogen
11. Pyrazole 10.66 2.105 5.0 (4.92) Weak carcinogen
12. Nitrosopyrrolidine 10.85 3.818 5.6 (5.72) Carcinogen
13. Dimethylnitrosamine 10.87 3.371 5.1 (5.18) Carcinogen
14. Halothane 10.94 1.730 5.6 (5.69) Weak carcinogen
15. Carbon tetrachloride 11.26 0.005 5.5 (5.52) Weak carcinogen
16. Acetone 11.51 2.896 4.9 (4.92) Non-carcinogen
17. Diethylether 13.38 1.247 5.4 (5.52) Non-carcinogen
18. Acetonitrile 14.13 2.893 4.4 (4.38) V. weak carcinogen
19. Propan-2-01 14.39 1.617 5.0 (5.14) Non-carcinogen
20. Ethanol 14.44 1.550 4.5 (4.64) V. weak carcinogen
21. Butan-2-01 14.51 1.624 5.4 (5.56) Non-carcinogen
22. Pentane 14.89 0.007 5.6 (5.74) Non-carcinogen

AE= E(LUMO)-E(HOM0) where: E(HOMO), E(LUM0) represent the energies (eV) of the highest occupied and lowest
unoccupied MOs; p, dipole moment (Debye); d, molecular diameter (A) where data in parentheses were calculated from the
solvent-accessible surface volumes.

oxidative metabolism of chemicals both endoge- In addition to their role in activating metabolic
nous and exogenous (Nelson et al., 1993). Al- pathways, CYP2E enzymes also mediate (Ronis et
though generally associated with detoxifying al., 1996; Terelius et al., 1993) the generation of
reactions, it is known that certain P45Os mediate oxygen radicals (e.g. 0,. and OH.) and other
the metabolic activation of procarcinogens, of reactive oxygen species (ROS), which represent
which the CYPlA and CYP2E subfamilies appear potential toxic agents (Kukielka and Cederbaum,
to be the major activating enzymes of xenobiotic 1994; Halliwell and Gutteridge, 1990). ROS also
compounds in man (Gonzalez and Gelboin, 1994). participate in the initiation and/or progression of
Substrates and inducers of CYP2E include a sub- cancer and various other degenerative disease
stantial number of known rodent (and suspected states; indeed, ROS generation may be linked with
human) carcinogens, such as: benzene, short-chain the process of ageing itself (Orr and Sohal, 1994).
alkyl nitrosamines, small molecular weight Apparently, the production of superoxide radicals,
haloalkanes, haloalkenes and certain alkenes (Ro- which has been associated with CYP2E (Gonder et
nis et al., 1996; Terelius et al., 1993; Raucy et al., al., 1985) may represent a mechanism for oxygen
1993; Nakajima et al., 1993; Yang et al., 1990; toxicity (Suzuki and Ford, 1993) resulting from
Guengerich et al., 1991). Consequently, a chemical exposure to chemical agents, such as those used as
which is both an inducer of CYP2E and activated general anaesthetics (Liu et al., 1993), or during
by the enzyme to a more reactive species may prolonged fasting (Yang et al., 1990). It is also
represent a significant toxic hazard to the organ- thought that the diabetic state can lead to oxygen
ism, as detoxifying metabolic pathways and DNA toxicity via enhancement of CYP2E activity, pre-
repair mechanisms could become overwhelmed. sumably associated with the production of ketone
 D.F.V. Lewis et al. 1 Toxicology 118 (1997) 93-113 95

Table 2
QSARs in series of CYPZE substrates

QSAR expression n SE R F

A. Nitriles (Lewis et al., 1994a,b)

1. -log Ki = -1.25a/AE+0.95 13 0.282 0.76 15.3
2. log EtOH/glucose = -0.006a+O.277 20 0.046 0.79 30.5
3. -logLD,, = 0.03~ f0.43 16 0.199 0.87 42.1
4. -logLD,, = 0.69o1/AE + 0.47 16 0.199 0.87 42.4
5. a = 15.97 diam-61.74 20 2.520 0.97 275.1
Ki = inhibition constant of nitrile metabolism following ethanol treatment
EtOH/glucose = fold increase in ethanol metabolism with respect to glucose
LD,, = lethal dose for 50% of animals treated
OL= calculated molecular polarizability (A3) using COSMIC
AE = E(LUM0) - E(HOM0) calculated by the CND0/2 method
diam = collision diameter (A) calculated via COSMIC

B. Alcohols (Lewis, 1987)

1. pIC,, = 0.45 length -4.04 22 0.636 0.74 23.6
2. plC,, = 13.3 Q,L+6.42 20 0.337 0.92 93.0
pIC,, = -log,,, concentration causing 50% inhibition of aniline p-hydroxylation
length = molecular length (A)
QoL = LUMO electron density on the r-carbon

C. Nitrosamines (Lewis et al., 1996)

1. - logLD,, = 4.79AE - 47.47 6 0.334 0.95 35.7
2. -logLD,, = 67.94, (HOMO)+3.57 25 0.327 0.84 51.6
LD,, = dose required to produce tumours in 50% of exposed rats
AE = E(LUM0) - E(HOM0) calculated via the CND0/2 method
Q,(HOMO) = HOMO electron density on the cc-carbon atom

D. Pyrazines (Lewis, 1996a,b)

area /depth ’
-logLD,, = 8.80 AE -4.95 5 0.087 0.94 15.2

LD,, = lethal dose in 50% animals treated

area/depth* = the quotient of molecular area and square of molecular depth
AE = E(LUM0) - E(HOM0) calculated via the CND0/2 method

n, no. of observations; SE, standard error; R, correlation coefficient; F variance ratio.

bodies, which are known to increase CYP2E ac- nisms between species for dealing with ROS pro-
tivity (Ioannides et al., 1995). There is, moreover, duction (Parke, 1987, 1990; Parke and Ioannides,
a link between body size, metabolic rate and 1990).
generation time in different species (Martin and
Palumbi, 1993) which indicates that smaller ani- 1.2. CYP2E characteristics
mals experience relatively short life-spans due to
their increased metabolic rate and, hence, greater It has been shown that, although structurally
oxygen consumption. Furthermore, there is a sim- diverse, CYP2E substrates all possess relatively
ilar relationship between the physical size of di- small molecular structures with collision diame-
verse animal species, induction time for diethyl- ters of 6.5 A or less (Lewis et al., 1994a,b) and
nitrosamine-induced tumour formation, and over- that, to some extent, the more toxic and carcino-
all life-span (Lijinsky, 1993), which indicates a genic CYP2E substrates tend to have relatively
common role for CYP2E enzymes, in addition to low activation energies as measured by the AE
the variation in natural biological defence mecha- value [AE = E(LUM0) - E(HOMO)], where
96 D.F.V. Lewis et al. / Toxicology
8 9 10
Fig. 1, A scatter plot of dipole moment (,u) versus the difference
inducers (data given in Table 1).
E(LUM0) and E(HOM0) represent the lowest
unoccupied and highest occupied molecular orbit-
al energy levels, respectively. Table 1 lists these
characteristics for a number of known CYP2E
substrates and inducers where it can be seen that,
by and large, the carcinogenic or hepatotoxic
CYP2E substrates possess low AE values corre-
sponding to a relative ease of activation. The
CYP2E substrates or inducers with relatively high
values of AE tend to be ROS generators and may,
therefore, be able to give rise to toxicity via this
mechanism. As the heme iron of CYP2E is natu-
rally in the high-spin state, this characteristic,
which may be unique in comparison to other
P45Os, facilitates the activation of oxygen even in
the absence of substrate (or if the inducer is a
poor substrate) thus producing superoxide anion
and, subsequently, hydroxyl radicals and other
ROS.
118 (1997) 93-113
11 12 13 15
Delta E
between frontier orbital energies (AE) for 22 CYPZE substrates and
1.3. QSARs for CYP2E substrates
The AE parameter features in a number of
QSAR expressions relating to the toxicity of vari-
ous CYP2E substrates, as shown in Table 2,
where there is some variation in the actual struc-
tural descriptors, depending on the type of activ-
ity and nature of the substrates. However, it can
be shown that some of the structural parameters
are interrelated. For example, the collision diame-
ter factor, which appears to discriminate CYP2E
specificity from that of other P45Os, correlates
closely (r = 0.97) with the molecular polarizability
parameter (a) that is involved in the QSARs for
nitriles (Table 2). Furthermore, the molecular
length characteristic, which correlates with the
CYP2E inhibitory activity of aliphatic alcohols, as
shown in Table 2, is essentially related to the
molecular diameter, as there is a fairly good inter-
 D.F. V. Lewis et al. / Toxicology 118 (1997) 93-l 13 91

Table 3
QSAR evaluations on a series of epoxides (Lewis, 1996a,b)

QSAR expression n SE R F

1. I%= 1339.1 E,/V,,,+0.99 9 12.19 0.93 48.22

2. 1% = 1230.2 E,/V,,,+6.37 log P-O.32 9 12.05 0.95 25.22
3. log 1% = 12.04 E,/V,,,+ 1.19 6 0.116 0.93 25.95
4. log I%= 11.21 E,/V,,,+O.ll log P+1.12 6 0.085 0.95 26.77

I%, percentage inhibition of epoxide hydrolase (Politzer and Laurence, 1984); E,, Taft steric substituent parameter (Politzer and
Laurence, 1984); V,,,, value of minimum electrostatic potential energy (Politzer and Laurence, 1984); log P, calculated values of log
P octanol/water using the Rekker method.

relationship (Y = 0.82) between these two structural As both dipole moment and polarizability are
parameters for the CYP2E-specific chemicals pre- two of the major components to log P, the hydro-
sented in Table 1. It can be seen from Table 1 that phobicity/lipophilicity parameter, it is possible that
some of the compounds are relatively polar, in that the log P value may be a useful descriptor for
they possess high dipole moments. Moreover, a estimating the potential toxicity of CYP2E sub-
plot of polarity versus AE for these chemicals (Fig. strates. Indeed, for a series of small molecular
1) indicates that the more carcinogenic CYP2E weight haloalkanes, the polarizability relates well
substrates combine low dipole moments with low (r = 0.86-0.88) with both experimental and calcu-
AE values, and it is possible that such compounds lated log P values (Lewis, 1995a). In this series of
are relatively lipophilic and may be poor substrates CYP2E substrates, polarizability is the major com-
for the Phase II conjugating enzymes. ponent of log P, but other classes of CYP2E-spe-
cific chemicals are more polar, in which case the
dipole moment makes an important contribution
to the overall log P (Lewis, 1989); and the magnit-
ude of E(HOM0) also plays a role in examples
where electron-donating capability relates to hy-
drogen bonding properties. Indeed, a combination
of polarizability, dipole moment and E(HOM0)
gives a good correlation (r = 0.91) with log P for
many structurally diverse small molecular weight
compounds (Lewis, 1989).
Table 2 shows that AE is a factor in QSARs for
nitriles, pyrazines and nitrosamines, many of
which are CYP2E substrates, and it should be
recalled that this parameter is the difference be-
4
tween the two frontier orbital energies. In some
monoepoxide
3-butenal cases, it is found that one or other frontier orbital
4
tautomelism is primarily related to metabolic activity. For
1 1 example, the relative rate of alkene epoxidation
(Traylor and Xu, 1988) appears to be dependent
oH y (r = 0.85) upon the ionization energy, which is
crotonaldehyde
diepoxide essentially the same as the E(HOM0) value.
4

butenedial ?
(Bird, et al., 1987)
However, the rates of base-catalyzed
of aliphatic ketones, a reaction which may mimic
detritiation

Scheme 1. Proposed mechanism of P450-mediated butadiene the P450 oxidative process, correlate fairly well
oxygenation, (r = 0.78) with E(LUM0). However, similar cor-
98 D.F.V. Lewis et al. / Toxicology 118 (1997) 93-113

Table 4
Similarities and differences between putative active residues in rat, mouse and human CYP2EI

Residue No. Rat Mouse Human Comments

72 Arg Arg Arg Ion-pairs with Asp-74

74 Asp Asp Asp Ion-pairs with Arg-72
78 Phe Phe Phe n-stacked with Phe-88
82 Lys Lys Arg May interact with some substrates
87” Ile Ile Ile Hydrophobic interaction with some substrates
88 Phe Phe Phe n-stacked with Phe-78
181a Phe Phe Phe n-stacking with some substrates
182 Tyr Tyr His May restrict access to active site
185 Ser Ser Ser Possible hydrogen bond donor
263 Phe Phe Phe n-stacking with Phe-181 and restriction of active site
266 Thr Thr Thr May interact with some substrates (Fukuda et al., 1993)
267” GlU Glu Glu May interact with some substrates
328” Val Val Val Hydrophobic interaction with some substrates
331” Asn Asn Asn May restrict size of active site and interact with substrates
436 Val Val Ile May relate to species differences in metabolism
437” Thr Thr His May interact with some substrates
438 Val Ile Ile May relate to species differences in metabolism

“Residue positions which have been identified via site-directed mutagenesis as being important for substrate regio- and stereo-selec-
tivity in other CYP2 family proteins (Lewis, 1995b).

relations occur between E(LUM0) and rate con- and hydrophobic, possessing fairly high log P
stants (r = 0.71-0.83) for the nitration of mono- values. Such chemicals are likely to be more resis-
substituted benzene derivatives, which include tant to oxygenation via CYP2E although they
many CYP2E substrates. In fact, such relation- may enhance the enzyme activity, giving rise to
ships may explain why aniline is para-hydroxy- ROS (as in the case of benzene), and could also
lated by CYP2E, whereas p-nitrophenol is form epoxides if they are unsaturated hydrocar-
hydroxylated meta to the nitro group. It is found bons (e.g. buta-1,3-diene). If these epoxides are
that although both meta- and ortho-substitution themselves poor substrates for epoxide hydrolase
rates are proportional to E(LUMO), the rate of (EH) then they may be carcinogenic via DNA
para-substitution correlates closely (Y = 0.91) with alkylation, as the epoxide carbon atoms tend to
the electrophilic superdelocalizability of the ring be strongly electrophilic and, therefore, attack the
carbon atom in the para position. highly nucleophilic DNA bases such as guanine.
Many CYP2E substrates are fairly polar, with Consequently, if CYP2E substrates possess
relatively high dipole moments (Table 1) which structural characteristics which predispose them
lead, therefore, in some cases, to low values of log to form epoxides (e.g. benzene, butadiene, iso-
P, as dipole moment can represent a significant prene, ethene and its chlorinated derivatives), then
contribution to the overall log P value for rela- their potential toxicity is likely to depend upon
tively polar compounds (Lewis, 1989). It is likely their relative ease of hydration by EH. It is possi-
that both polarity and hydrogen bond potential ble to determine the strength of interaction with
are important in interactions between CYP2E EH via QSAR analysis of known epoxides.
substrates and critical amino acid residues in the Politzer and Laurence (1984) have shown that
enzyme active site, where dipolar forces (including there is a relationship between EH interaction and
hydrogen bonding) are probably involved in sub- molecular electrostatic potential (MEP) energy
strate recognition and orientation for metabolism minima of the epoxides themselves, thus indicat-
in the known positions. However, some CYP2E ing the possibility of hydrogen bond formation
substrates and inducers are relatively non-polar between the epoxide substrate and an active site
 D.F. V. Lewis et al. 1 Toxicology 118 (1997) 93-l 13 99

ALIGNMENT BETWEEN CYP102 AND CYPZE SUBFAMILY MEMBERS

10 20
cyp102 . .. . . . . . . . .. . . . .. . . . . . . . . . ..TI KEMPQPKTFG ELKNLPLLNT
cyplelhum MSALGVTVAL LVWAAFLLLV SMWRQVHSSW NLPPGPFPLP IIGNLFQLEL
cyp2elrab MAVLGITVAL LGWMVILLFI SVWKQIHSSW NLPPGPFPLP IIGNLIQLDL
cyp2elrat MAVLGITIAL LVWVATLLVI SIWKKIYNSW NLPPGPFPLP ILGNIFQLDL
cyp2elmou MAVLGITVAL LVWIATLLLV SIWKQIYRSW NLPPGPFPIP FFGNIFQLDL

30 40 50 60 70
cyplO2 DKPVQALMKI ADELGEIFKF EAPGRVTRYL SSQRLIKEAC DESRFDKNLS
cyplelhum KNIPKSFTRL AQRFGPVFTL YVGSQRMVVM HGYKAVKEAL LDYKDEFSGR
cyp2elrab KDIPKSFGRL AERFGPVFTV YLGSRRVVVL HGYKAVREML LNHKNEFSGR
cyplelrat KDIPKSFTKL AKRFGPVFTL HLGSRRIWL HGYKAVKEVL LNHKNEFSGR
cyp2elmou KDIPKSLTKL AKRFGPVFTL HLGQRRIWL HGYKAVKEVL LNHKNEFSGR

100 110 120

cyplO2 QALKFVR:;A GDGLFTS::H EK NWKKAHNI LLPSFSQQAM .K.GYHAHUV
cyp2elhum GDLPAFHAHR DRGIIFN..N GPTWKDIRRF SLTTLRNYGM GKQGNESRIQ
cyplelrab GEIPAFREFK DKGIIFN..N GPTWKDTRRF SLTTLRDYGM GKQGNEDRIQ
cyp2elrat GDIPVFQEYK NKGIIFN.. N GPTWKDVRRF SLSILRDWGM GKQGNEARIQ
cyp2elmou GDIPVFQEYK NKGIIFN..N GPTWKDVRRF SLSILRDWGM GKQGNEARIQ

130 140 150 160 170

cyp102 DIAVQLVQKW ERLNADEHIE VPEDMTRLTL DTIGLCGFNY RFNSFYRDQP
cyp2elhum REAHFLLEAL RKTOG.OPFD PTFLIGCAPC NVIADILFRK HFDYNDEKF.
cyp2elrab KEAHFLLEEL RKTGG.iPFD PTFVIGCTPF NVIAKILFND RFDYKDKQA.
cyp2elrat REAQFLVEEL KKTKG.QPFD PTFLIGCAPC NVIADILFNK RFDYNDKKC.
cyp2elmou REAHFLVEEL KKTKG.QPFD PTFLIGCAPC NVIADILFNK RFDYDDKKC.

180 190 200 210

cyp102 HPFITSMVRA LDEAMNKLQR A..NPDDP.. AYDENKRQFQ EDIKVMNDLV
cyp2elhum LRLMYLFNEN FHLLSTPWLQ LYNNFPSFLH YLPGSHRKVI KNVAEVKEYV
cyplelrab LRLMSLFNEN FYLLSTPWLQ VYNNFSNYLQ YMPGSHRKVI KNVSEIKEYT
cyplelrat LRLMSLFNEN FYLLSTPWIQ LYNNFADYLR YLPGSHRKIM KNVSEIKQYT
cyp2elmou LELMSLFNEN FYLLSTPWIQ AYNYFSDYLQ YLPGSHRKVM KNVSEIRQYT

220 230 240 250 260

cyp102 DKIIADRKAS GEQS..DDLL THMLNGKDPE . ..TGEPLDD ENIRYQIIT
cyplelhum SERVKEHHQS LDPNCPRDLT DCLLVEMEKE KHSAERLYTM DGITVTVAD
cyplelrab LARVKEHHKS LDPSCPRDFI DSLLIEMEKD KHSTEPLYTL ENIAVTVAD
cyplelrat LEKAKEHLQS LDINCARDVT DCLLIEMEKE KHSQEPMYTM ENVSVTLAD
cyp2elmou LGKAKEHLKS LDINCPRDVT DCLLIEMEKE KHSQEPMYTM ENISVTLAD

270 280 290 300

cyp102 FLIAGHETTS GLLSFALYFL VKNPHVLQKA AEEAARVLV. DPVPSYKQVK
cyplelhum LFFAGTETTS TTLRYGLLIL MKYPEIEEKL HEEIDRVIGP SRIPAIKDRQ
cyp2elrab MFFAGTETTS TTLRYGLLIL LKHPEIEEKL HEEIDRVIGP SRMPSVRDRV
cyp2elrat LFFAGTETTS TTLRYGLLIL MKYPEIEEKL HEEIDRVIGP SRVPAVRDRL
cyplelmou LFFAGTETTS TTLRYGLLIL MKYPEIEEKL HEEIDRVIGP SRAPAVRDRM

310 320 330 340 350

CYD102 QLKYVGMVLN EALRLWPTAP AlFSLYAKED TVLGGEYPLE KGDELMVLIP
cypleihum EMPYMDAWH EIQRFITLVP SNLPHEATRD TIFRG.YLIP KGTVVVPTLD
cyp2elrab QMPYMDAWH EIQRFIDLVP SNLPHEATRD TTFQG.YVIP KGTWIPTLD
cyp2elrat DMPYMDAWH EIQRFINLVP SNLPHEATRD TVFQG.YVIP KGTVVIPTLD
cyp2elmou NMPYMDAWH EIQRFINLVP SNLPHEATRD TVFRG.YVIP KGTWIPTLD

360 370 380 390 400

cyplO2 QLHRDKTIWG DDVEEFRPER FENPSAI..P QHAFKPFGNG QRACIGQQFA
cyp2elhum SVLYDNQEFP .DPEKFKPEH FLNENGKFKY SDYFKPFSTG KRVCAGEGLA
cyplelrab SLLYDKQEFP .DPEKFKPEH FLNEEGKFKY SDYFKPFSAG KRVCVGEGLA
cyp2elrat SLLYDSHEFP .DPEKFKPEH FLNENGKFKY SDYFKAFSAG KRVCVGEGLA
cyp2elmou SLLFDNYEFP .DPETFKPEH FLNENGKFKY SDYFKAFSAG KRVCVGEGLA

410 420 430 440 450

cyp102 LHEATLVLGM MLKHFDFE.D HTNYELDIKE TLTLKPEGFV VKAKSKKIPLGG
cyp2elhum RMELFLLLCA ILQHFNLKPL VDPKDIDLSP IHIGFGC.IP PRYKLCVIPRS
cyp2elrab RMELFLLLSA ILQHFNLKPL VDPEDIDLRN ITVGFGR.VP PRYKLCVIPRS
cyp2elrat PJlELFLLLSA ILQHFNLKSL VDPKDIDLSP VTVGFGS.IP PQFKLCVIPRS
cyp2elmou RMELFLLLSA ILQHFNLKSL VDPKDIDLSP VTIGFGS.IP REFKLCVIPRS
Fig. 2. An alignment between the amino acid sequences of CYP102 and members of the CYPZE subfamily produced using the GCG
package and modified to conform with the multi-family alignment (Lewis, 1995b).
100 D.F. V. Lewis rt al. ! Taxicohgv I18 (19971 93-113

Fig. 3. A view of the entire human CYP2El model showing the location of the region recognized by a specific antibody to CYP2E
(green), basic residues (dark blue) likely to form ion-pairs with the heme propionates, and the putative active site residues (red). The
hcmc moiety is shown in magenta.
Fig. 4. A view of the putativr active site of the mouse CYPZEI orthologue. showing the likely orientation of butadiene epoxide
within the heme environment. Hydrogen bonds are displayed as dashed lines.
 D.F.V. Lewis et al. / Toxicology 118 (1997) 93-113 101

Fig. 5. Stereo-pair of the putative active site of rat CYPZEl, showing a possible orientation of the specific substrate p-nitrophenol,
where dashed lines indicate hydrogen bond interactions with amino acid residues in the heme pocket.

amino acid residue in EH. It can be demonstrated although CYP2E-mediated carcinogenicity is as-
that a simple transformation of the original data sociated with relatively low values of AE, the
generated by Politzer and Laurence (1984) to- presence of a high dipole moment in the molecule
gether with the inclusion of log P values (as an tends to reduce the likelihood of activation. There
estimate of hydrophobicity), provides a useful also appears to be an optimum range of molecular
QSAR expression (Table 3) for the prediction of diameters (between 5.0 and 6.0 A) and AE values
EH-mediated toxicity. As the rate of P450-medi- (between 9.0 and 11.5 eV) where the more carcin-
ated epoxidation of alkenes (and, presumably, ogenic CYP2E substrates tend to cluster (Fig. 1).
aromatics) is found to be proportional to It is possible, however, that most CYP2E sub-
E(HOM0) or, more precisely, ionization energy strates and inducers have the potential for car-
[where: IE = - E(HOMO)], it should be possible cinogenicity or hepatotoxicity, especially in small
to assess the likely toxicity of these compounds rodents, as poor substrates possess the propensity
via a combination of the two QSAR expressions, for ROS generation; the outcome of which will
i.e. one for the formation of the epoxide and the depend on many other factors including: species,
other for the resulting carcinogenic potential of sex, diet, exposure, environment, dose regimen
the epoxide formed. and genetics.
However, not all CYP2E-mediated toxicity in- The relationship between E(HOM0) and rate
volves epoxidation and, for example, mutagenicity of epoxidation has been mentioned previously,
can be dependent upon E(LUMO), although this and it is possible that the AE parameter may
process may not necessarily be a P450-activated encompass a variety of CYP2E-mediated activa-
reaction. Nevertheless, the use of AE, which in- tion mechanisms, as this quantity could represent
cludes both E(LUM0) and E(HOMO), may rep- a contribution to the overall activation energy of
resent a means for discriminating between the P450 oxygenation reaction (Lewis, 1995a);
carcinogens and non-carcinogens. Coupled with reduction is also possible in the case of haloalkane
the dipole moment factor, this appears to be quite metabolism. Although the connection between the
useful for differentiating carcinogens from non- thermodynamics of substrate binding and kinetics
carcinogens in CYP2E substrates, as can be ap- of P450-mediated reactions has yet to be fully
preciated from Fig. 1. This analysis suggests that, elucidated, it is likely that compound hydropho-
 D.F. V. Lewis et al. / Toxicology 118 (1997) 93-l 13

Fig. 6. Stereo-pair of the rat CYP2EI active site model with chlorzoxazone orientated for metabolism at the known position.

bicity plays an important role in the energetics of substrate interactions. Nevertheless, hydrophobic-
binding to various P450-isozymes (White and Mc- ity plays the dominant role in both desolvation of,
Carthy, 1986). In particular, desolvation of the and binding to, the P450 active site and, conse-
enzyme active site which accompanies substrate quently, log P or its major component, namely,
binding brings about a significant, favourable en- the molar polarizability, CI,,, (Lewis, 1989), will
tropy change that represents a major component represent important structural descriptors for
of the overall binding energy (Griffin and Peter- P450-substrate interactions.
son, 1972). Furthermore, it has been shown that In many instances, the molar volume, V, and
the volume of the solvent-accessible surface (V) log P are related, as is molecular diameter and
correlates closely (Y = 0.97) with the free energy of solvent-accessible volume or surface area, so any
binding for a series of p-substituted toluenes bind- of these factors can be useful in quantifying the
ing to CYP2B4 (Lewis et al., 1993, whereas the degree of interaction between P450 isozymes and
hydroxylation rate of these compounds exhibits a their substrates. Clearly, being a component of
parallelism (v = 0.98) with a combination of V log P, dipole moment is also likely to figure in
and dipole moment (Lewis et al., 1993, thus QSAR expressions relating to P450-substrate
indicating that there may be a link between the binding processes, with AE or one of its compo-
thermodynamics of binding to, and rate of reac- nent frontier orbital energies representing a third
tion by, P450 (Scheme 1). structural descriptor of potential relevance to
In the case of the bacterial form P450,,, isoenzyme specificity and either charge-transfer
(CYPlOl) obtained from Pseudomonas putidu, a interactions or activation propensity. The data
hydrogen bond between an active site tyrosine presented in Table 1 and Fig. 1 show that this is
residue (Tyr-96) and the carbonyl group of the indeed the case for CYP2E substrates and induc-
camphor substrate helps to anchor the molecule ers.
in the heme environment for hydroxylation, al-
though several hydrophobic contacts orientate the 1.4. Modelling of CYP2El isozymes
substrate for stereo-specific hydroxylation at the
5-exo position (Poulos et al., 1987). As there is As part of an extensive analysis of P450 sub-
likely to be a major dipolar component to the strates and their respective isozyme specificities,
hydrogen bond interaction, substrate dipole mo- with respect to both toxicity and metabolism,
ment will probably be a factor in both P450 various mammalian P45Os (Lewis, 1995b; Lewis
binding and isozyme specificity for other enzyme- and Lake, 1995) have been constructed from a
 D.F. V. Lewis et al. 1 Toxicology I I8 (1997) 93-l 13

Fig. 7. Aniline is shown docked into the putative active site of rat CYP2El where key amino acid side chains orientate the substrate
for p-hydroxylation.

recent bacterial crystal structure (CYP102) which (Bond et al., 1995), whereas mechanistic data
represents a preferred structural template for eu- suggest that it is a human carcinogen (Melnick
karyotic P45Os (Ravichandran et al., 1993). The and Kohn, 1995). There is evidence that butadiene
present work is concerned with modelling of is activated by CYP2E isozymes (Melnick and
CYP2El isozymes from rat, mouse and human to Kohn, 1995), and it was considered that a study
investigate substrate interactions and, in particu- of the active sites of mouse, rat and human
lar, to attempt rationalization of species differ- CYP2E isozymes, and of their differences, might
ences in butadiene metabolism (Duescher and explain the differences in the rodent activation of
Elfarra, 1994; Csanady et al., 1992) that may butadiene, and enable the potential for carcino-
relate to altered carcinogenicity profiles between genicity in human to be more scientifically evalu-
these species (Bond et al., 1995; Melnick and ated.
Kohn, 1995).

1.5. Butadiene toxicity 2. Methods

1,3-Butadiene, a minor constituent of gasoline The models of CYP2El enzymes for the rat,
and diesel fuel, a toxic component of automobile mouse and human orthologues were constructed
emissions, and an industrial chemical used in high from the CYP102 crystal structure (Ravichandran
tonnage in polymer manufacture, is carcinogenic et al., 1993) using a multiple sequence alignment
in mouse but not in rat, data that has caused between the relevant protein sequences, as shown
concern for the potential carcinogenic risk to man in Fig. 2. The underlying principles governing this
from exposure to butadiene (Bird et al., 1987; form of sequence alignment have been discussed
Csanady et al., 1992). It is known that butadiene previously (Lewis, 1995b). Four bacterial P450
is converted to its mono-epoxide in both mouse crystal structures have now been determined
and rat, but that the mono-epoxide is activated to (Hasemann et al., 1995; Cupp-Vickery and Pou-
the di-epoxide, the proximate carcinogen, only in los, 1995) and are shown to display a generally
mouse but not in rat (Bond et al., 1995). conserved tertiary fold, with some significant dif-
Epidemiological studies have indicated that bu- ferences within the substrate binding sites, espe-
tadiene, at workplace environmental concentra- cially with respect to the loop regions following
tions, would not be carcinogenic to humans the B’ and F helices (Hasemann et al., 1995).
 104 D.F. V. Lewis et al. / Toxicology 118 (1997) 93~ 113

Fig. 8. The substrate, paracetamol, is shown in the rat CYP2EI active site model and orientated for me/a-hydroxylation

Several aspects of the CYP102 bacterial form butadiene epoxide and diallyl sulphide.
render it the most suitable template structure for
generation of mammalian microsomal P45Os
(Lewis, 1995b). These include the fact that it is of 3. Results
approximately the same length in terms of amino
acid sequence as eukaryotic P45Os; apart from the The sequence homology exhibited by the multi-
N-terminal anchor region, it is of generally closer ple alignment (Fig. 2) is 17% in terms of overall
homology than the other bacterial P450 crystal identity between the four CYP2El proteins and
structures, and it shares a common redox partner CYP102, whereas the conservation shown in this
in NADPH-dependent cytochrome P450 reduc- alignment is 35%. For individual CYP2E proteins
tase, whereas the other prokaryotic forms all re- these percentages increase to 25% homology and
quire an iron-sulphur redoxin as a source of as high as 54% conserved residues in some cases,
reducing equivalents (Lewis, 199513). whereas the homology between, for example, rab-
Each CYP2El structure was generated by bit CYP2El and CYPlOl is significantly lower at
amino acid replacement as required by the se- 17X, thus indicating that CYP102 represents a
quence alignment (Fig. 2) using the Biopolymer preferred template to CYPlOl. In particular, the
module of the Sybyl molecular modelling software C-terminal portion (from the I helix onwards) is
(Tripos Associates, St. Louis, MO). Following the considerably better conserved than that of the
deletion and insertion of the small number of N-terminus, as has been noted in ‘across-family’
amino acid residues required by the modelling alignments (Lewis, 1995b; Hasemann et al., 1995).
alignment, the raw structure in each case was Only eight residue deletions were required by the
energy minimized using the Tripos force field to modelling alignment shown in Fig. 2, together
obtain an optimized geometry, according to the with 16 residue insertions, all of which involved a
criteria described previously (Lewis, 1995b; Lewis small number of residues, in each case of no more
and Lake, 1995). The minimum energy geometries than three amino acids, as can be appreciated by
of the CYP2El enzymes were then probed via inspection of Fig. 2.
interactive docking using the previously mini-
mized structures of known substrates and in- 3.1. CYP2E mobculur models
hibitors of the CYP2E subfamily including:
p-nitrophenol, ethanol, acetone, dimethylni- The resultant geometries of the CYPZE models
trosamine, aniline, chlorzoxazone, disulphiram, consequently minimized smoothly over several
 D.F.V. Lewis et al. / Toxicology 118 (1997) 93-113 105

Fig. 9. Ethanol is shown within the rat CYP2El model where contacts with active site residues orientate the substrate for
oxygenation via the heme moiety.

hundred iterative cycles to yield highly stable adduct studies (Mackman et al., 1996). In particu-
structures with internal energies of around lar, Phe-181, Phe-263, His-437 and Arg-82 side
- 1200 kcal mol ~ i. A substantial number of fa- chains, together with that of Asn-331, line the
vourable contacts between complementary amino putative substrate binding site, in agreement with
acid residues are observed in the minimized the substrate recognition sites (SRSs) proposed by
CYP2E structures, including electrostatic ion- Gotoh (1992), such that it is possible for only
pairs, hydrogen bonds and hydrophobic interac- small-sized molecules to enter the heme pocket.
tions (Table 4 and Fig. 3) some of which appear Complementarity between these amino acid
to be common to all mammalian microsomal residues and substrates probably explains the en-
P45Os, such as those associated with the binding zyme specificity towards either small molecular
of the heme moiety and with redox partner inter- weight aromatic compounds or small polar
actions (Lewis, 1995b; Lewis and Lake, 1995). molecules with hydrogen bond potential. It is
Consequently, although the relevant enzyme possible that Thr-266 on the I helix is able to
crystal structure would be preferable, a model enter into hydrogen bonded interactions with
based on homology with an analogous crystal many CYP2E substrates, and other candidate hy-
structure template represents a reasonable alterna- drogen bonding residues include Arg-82, Asn-33 1
tive for the investigation of substrate binding and possibly His-437 (Table 4). As the latter
interactions. becomes Thr in both rat and mouse CYP2E, this
Fig. 4 shows the structure of the entire CYP2E fact together with a number of other changes
model, where the region recognized by specific within the putative active sites (Table 4) may
antibodies (Edwards et al., 1991) is indicated, explain some of the species differences in CYP2E-
together with the putative active site. The sub- mediated metabolism.
strate binding pocket appears to be bounded by a
number of relatively bulky amino acid residues
which restrict the overall size such that only rela- 4. Discussion
tively small molecules are able to enter the heme
environment, and this is in agreement with the 4.1. Species differences in C YP2E active sites
known substrate specificity of CYP2E enzymes
(Koop, 1992; Hargreaves et al., 1994; Yang et al., A current interest in the species variation in
1990) together with recent evidence from heme butadiene metabolism has been a major factor in
106 D.F. V. Lewis rt al. / Toxicology 118 (1997) 93-l 13

Fig. 10. The CYP2El substrate, acetone, is shown docked into the rat CYP2El active site where hydrogen bonding and other
interactions orientate the substrate for methyl oxygenation.

generating models of rat, mouse and human within the three mammalian species. In particular,
CYP2E, as it is known that activation of the His-437 in human CYP2E becomes Thr-437 in
mono-epoxide of butadiene to its diepoxide oc- both rat and mouse, whereas His-182 in the hu-
curs in the mouse but not in rat (Bond et al., man orthologue changes to Tyr-182 in the two
1995), and in man, the activation pathway is rodent forms. Furthermore, the two hydrophobic
similar to that shown in the mouse (Csanady et residues flanking His-437 in human CYP2E ex-
al., 1992). The fact that butadiene is carcinogenic hibit minor, but possibly significant, alterations in
in mouse but not in the rat has prompted concern the rat and mouse enzymes. The residue preceding
regarding the potential carcinogenic risk to man His-437 is Be-436 in the human orthologue but
from exposure to butadiene (Melnick and Kohn, becomes valine in both species of rodent and this
1995; Duescher and Elfarra, 1994). However, change represents the loss of a -CH,- group,
there is some evidence to suggest that CYP2A thus making the site somewhat open in this re-
isozymes may also be involved in butadiene acti- gion. The residue following His-437 in human
vation (Duescher and Elfarra, 1994). If this repre- CYP2E is also isoleucine (Ile-438) and is the same
sents a major pathway in mammalian species, in the mouse, but again changes to valine in the
then it is possible that the more marked differ- rat (Table 4). The overall effect of modifications
ences in the putative active sites of mouse, rat and within this region of the CYP2E active site (i.e.
human CYP2A isoforms (Lewis and Lake, 1995) residues 436-438) is likely to bring about a small,
may provide an alternative explanation for the but perhaps important, increase in the size of the
significant species difference between butadiene substrate binding pocket from human to mouse
carcinogenicity in rat and mouse. It is also inter- and then to rat as the amino acid residues change
esting to note that two of the putative active site from Be-His-Be (human) to Val-Thr-Be (mouse)
residues (Phe-181 and His-437) in human CYP2E and then to Val-Thr-Val (rat) for positions 436-
are the same in CYP2A6, which is the human 438.
orthologue (Lewis and Lake, 1995). Site-directed mutagenesis studies have shown
Nevertheless, there are several notable differ- that the analogous region in CYP2B is likely to
ences between the putative active sites of rat, contact substrates, whereas the previously men-
mouse and human CYP2E which could possibly tioned Phe-181 has been demonstrated as relevant
explain the variation in butadiene metabolism to substrate specificity in CYP2A orthologues;
 D.F. V. Lewis et al. / Toxicology 118 (1997) 93-113 107

Fig. 11. Dimethylnitrosamine is displayed within the rat CYP2El model, and orientated for methyl hydroxylation via hydrogen
bond interactions with active site residues.

moreover Asn-331 occupies a position corre- increased di-epoxidation of butadiene in the

sponding to a known allelic variant site in rabbit mouse whereas, in the rat, the mono-epoxide
CYP2C3 which gives rise to substrate regioselec- could diffuse out of the heme pocket more read-
tivity differences, and these findings have been ily. In human CYP2E, the change from threonine
reviewed recently (Lewis, 1995b; Lewis and Lake, to histidine at position 437 precludes hydrogen
1995). It would appear that histidine-437 (to- bonding to the oxygen of butadiene mono-epox-
gether with Phe-181) can assist in binding the ide and, therefore, it is likely that there will be a
planar butadiene substrate in human CYP2E, but different orientation of this metabolite for the
the change to threonine at position 437 in the second epoxidation step. Threonine-266 is a can-
rodent forms will facilitate hydrogen bonding to didate for hydrogen bond donation to the mono-
the mono-epoxide metabolite such that a second epoxide but the resulting change in substrate
epoxidation is possible. orientation could give rise to different metabolite
ratios relative to the situation in either rat or
4.2. Epoxidation of butadiene mouse, and these factors may have a bearing on
the carcinogenic potential of butadiene in the
The modelling indicates that a change from three mammalian species.
isoleucine to valine at position 438 may affect the It is known that initial oxygenation of butadi-
extent of di-epoxide formation, as the effect of the ene can give rise to either the mono-epoxide or
additional methylene group in the CYP2E active crotonaldehyde, and the relative amounts of these
site will tend to bind the mono-epoxide more primary metabolites may be dependent on the
strongly in the mouse enzyme relative to the topography of the CYP2E active site in the partic-
situation in the rat isoform, possibly leading to ular species in question. If this is the case, then
108 D.F. V. Lewis et al. I Toxicology 118 (1997) 93- 113

‘r”
28

Fig. 12. The specific inhibitor, disulphiram, is shown docked in the rat CYP2El active site where key amino acid residues position
the molecule for interaction with the heme group.

the ability of the enzyme to retain the mono-epox- and Phe-78, of which the latter two phenylala-
ide such that di-epoxidation is possible will be nines tend to form a coplanar pair of phenyl rings
important for determining the likely carcinogenic- at one edge of the putative active site of CYP2E.
ity of butadiene in that species. Although croton- Site-directed mutagenesis experiments have
aldehyde is a fairly toxic compound, one can been carried out on analogous positions (or adja-
assume that the two successive epoxidations of cent to them) in other CYP2 family proteins (see
butadiene represent the major activation pathway Lewis, 1995b for a summary) and have been
and, presumably, the orientation of the original shown to exhibit altered substrate regio- or stereo-
substrate (and metabolite) in the CYP2E active specificities. Consequently, it is likely that the
site will determine likely carcinogenic activation; aforementioned residues occupy the CYP2E ac-
the nature and disposition of active site amino tive site and participate in substrate binding and
acid residues are therefore, important determi- orientation for oxygenation. Together with those
nants of butadiene metabolism by CYP2E already mentioned, one or more of these amino
isozymes in different species. acid residues appear to be involved in the binding
It is possible, furthermore, that similar consid- of CYP2E substrates and inhibitors, as detailed in
erations are of relevance to CYP%E-mediated acti- the following section which relates to interactions
vation of other small molecular weight alkenes, with the rat CYP2E model.
although we have confined our investigation to
butadiene in the present study due to the current 4.3. Interactions between CYP2E substrates
interest in species differences in butadiene carcino- within the putative active site
genicity. However, as far as relatively planar hy-
drocarbons are concerned, there appears to be a The specific CYP2E substrate, p-nitrophenol, is
number of aromatic and hydrophobic aliphatic metabolized by the enzyme to form the 2-hydroxy
amino acid side-chains in the putative active sites 4-nitrophenol and, therefore, it is likely that the
of all three mammalian CYP2E enzymes which substrate molecule is orientated by the active site
may be of relevance to the access and binding of residues such that oxygenation occurs in the ortho
aromatic and olefinic substrates. These include: position with respect to the phenolic group. Fig. 5
Ile-87, Ala-264, Val-328, Val-438, Phe-181, Phe-88 shows how p-nitrophenol could bind to the puta-
 D.F. V. Lewis et al. / Toxicology 118 (1997) 93-l 13 109

Fig. 13. Diallyl sulphide, an inhibitor of the enzyme, is presented in the putative active site of rat CYP2E1, showing how the
structure may be orientated for heme interaction.

tive active site of rat CYP2E by forming hydrogen tation for n--71 stacking with the benzene ring of
bond interactions with the side-chains of the chlorzoxazone molecule, as is the case with
threonine-266 and threonine-437, together with another aromatic CYP2E substrate, aniline, which
n--72 stacking between phenylalanine-181 and the is preferentially hydroxylated in the para position.
aromatic ring of the substrate. Similarly, other In a similar way to chlorzoxazone, aniline is able
aromatic substrates can n-stack with the phenyl to form hydrogen bonds with Thr-266 and Glu-
ring of Phe- 18 1, although the side chain of Arg-82 267, but the side-chain of Thr-437 does not ap-
(Lys-82 in rat CYP2E) may be involved in hydro- pear to participate in hydrogen bonding with this
gen bonding to the nitro group of p-nitrophenol, substrate, as shown in Fig. 7.
especially in human CYP2E as Thr-437 becomes The presence of a number of aromatic residues
His-437 and, consequently, would be unable to in the CYP2E active site indicates that the protein
act as a hydrogen bond donor residue. However, may control the enzyme specificity for mono-aro-
in the rat form of the enzyme, Thr-437 appears to matic and other small molecular weight sub-
be able to enter into hydrogen bonded interac- strates, and the striking coplanarity of the phenyl
tions with most CYP2E substrates, e.g. chlorzoxa- rings of Phe-78 and Phe-88 indicates that this
zone, paracetamol, ethanol, acetone, dimethyl feature could represent an access channel for aro-
nitrosamine, and with the inhibitor, disulphiram, matic substrates. Although there is no evidence
although this compound is also able to inhibit from site-directed mutagenesis experiments that
CYP2A isozymes in addition to CYP2E. either of these phenylalanine residues participate
For example, chlorzoxazone is hydroxylated by in substrate interactions, the adjacent Ile-87 corre-
CYP2E in the 6-position which is ortho to the sponds to a position which has been investigated
chlorine substituent. Fig. 6 shows how this sub- in both the CYP2B and CYP2C sub-families, and
strate may be orientated in the putative active site is known to play a role in endogenous substrate
by interaction with Thr-437 and Thr-266 which, selectivity (Table 3). Moreover, this residue is
together with Glu-267, position the molecule such phenylalanine in CYP102 and has been shown by
that the known site of oxygenation lies directly site-directed mutagenesis to be involved in ensur-
above the iron-oxygen active centre. The phenyl ing regio-selectivity of lauric acid metabolism by
side-chain of Phe-18 1 is also in an optimal orien- preventing end-of-chain hydroxylation. Following
110

Fig. 14. A template of eight substrates of CYPZE (p-nitrophenol, chlorzoxazone, aniline, paracetamol, ethanol, acetone, dimethyl-
nitrosamine and butadiene epoxide) superimposed within the rat CYP2E model, where key interactions with active site amino acid
residues appear to be associated with substrate orientation for CYPZE-mediated metabolism.

the key position, Phe-181, on the F-helix which is tone and dimethylnitrosamine can all bind via
likely to represent a substrate contact in many Thr-437 which may orientate these compounds
P45Os, a section of peptide corresponding to for metabolism, and shown in Figs. 9- 11, respec-
residues 198-204 at the start of the G-helix con- tively.
stitutes an antibody recognition site in CYP2E For dimethylnitrosamine, the side-chain of Lys-
(Edwards et al., 1991). The current model indi- 82 is also likely to be involved in hydrogen bond-
cates that this region of peptide lies on the surface ing such that one of the methyl groups on the
of the protein and would, therefore, be readily substrate is positioned directly above the iron-
accessible to antibody binding. oxygen moiety. In some instances, hydrophobic
Although paracetamol can be metabolized by interactions are possible between these aliphatic
CYPl, it is also a known CYP2E substrate and is substrates and one or more of the side-chains of
hydroxylated ortho to the hydroxyl group in a the following amino acid residues, Ile-87, Ala-264,
similar manner to that of the specific CYP2E Val-328 and Val-438. As has been mentioned
marker substrate, p-nitrophenol. Fig. 8 shows a previously, butadiene and other structurally re-
possible orientation of paracetamol in the puta- lated small molecular weight olefins could become
tive active site of CYP2E, where a combination of orientated for oxygenation by interaction with
n-stacking with Phe- 18 1 and hydrogen bonding to these aliphatic residues, together with the possibil-
Thr-437, Thr-266 and Glu-267 positions the sub- ity of n-stacking to Phe-181. It is not known,
strate for metabolism at the experimentally ob- however, if the role of Asn-331 is to restrict the
served site. In addition to aromatic compounds, a size of the active site or whether the amide side-
number of small polar aliphatic molecules are also chain can hydrogen bond with some CYP2E sub-
CYP2E substrates. In these cases, Thr-437 is strates.
likely to be involved in hydrogen bonding with In addition to the substrates of CYP2E, known
complementary electronegative atoms on the sub- inhibitors such as disulphiram and diallyl sulphide
strates, and the CYP2E substrates, ethanol, ace- are also able to bind within the putative active site
 D.F. V. Lewis et al. / Toxicology 118 (1997) 93-l 13 111

by interacting with some of the same amino acid References

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