0090-9556/06/3401-1-7$20.00
DMD 34:1-7, 2006
MINIREVIEW
MECHANISM-BASED INACTIVATION AND REVERSIBILITY: IS THERE A NEW TREND IN THE INACTIVATION OF CYTOCHROME P450 ENZYMES?
Anna L. Blobaum
Department of Biochemistry, Vanderbilt University, Nashville,
Tennessee
(Received July 14, 2005;
accepted October 25, 2005)
 |
Abstract
|
|---|
Recent studies with cytochrome P450 (P450) enzymes from the 2E and 2B
subfamilies have shed light on what may be a new trend in the mechanism-based
inactivation of P450s: reversibility. The reversible inactivation of P450-type
enzymes was first reported in the mid-1990s by Dexter and Hager [Dexter AF and
Hager LP (1995
) J Am Chem
Soc 117:817–818], who studied the transient heme
N-alkylation of chloroperoxidase by allylbenzene and 1-hexyne. While
characterizing small tert-butyl acetylenes as mechanism-based
inactivators of P450s 2E1 and 2B4, Hollenberg and coworkers observed the
reversible inactivation of an acetylene-inactivated T303A mutant of P450 2E1.
The mechanism of reversibility was a combined product of the structure of the
inactivator and the positioning of conserved amino acid residues, threonine
303 (alanine in the mutant) and glutamate 302, in the enzyme active site.
Reversibility was also observed with both wild-type P450 2B4 and the T302A
mutant of 2B4, although this inactivation and reversibility did not seem to
depend on the T302 residue. Subsequent studies have attempted to elucidate the
chemical/structural requirements of the inactivator in determining
reversibility and have shown that both the size and the chemical nature of
functional groups play an important role. At this time, reversibility has only
been observed with P450 2E and 2B enzymes during their mechanism-based
inactivation by terminal alkynes. Future studies with P450s from other
subfamilies and structurally distinct inactivators will greatly aid our
understanding of the molecular and chemical determinants of reversibility.
|
Mechanism-Based Inactivation and Reversibility (Overview)
|
|---|
A wide variety of xenobiotic compounds have been shown to undergo metabolic
activation by the cytochrome P450 enzymes to form biologically reactive
intermediates that may, in turn, target the P450 for inactivation. The
specific binding of these reactive intermediates to the active sites of the
P450s has been exploited in the design of irreversible inhibitors, otherwise
defined as suicide or mechanism-based inactivators
(Massey et al., 1970;
Rando, 1984). Mechanism-based
inactivators can be broadly classified as substrates for the P450 that, in the
process of metabolism by the enzyme, are converted to a reactive intermediate,
which then irreversibly inactivates the enzyme without leaving the active site
(Silverman, 1995). There are
three different mechanisms by which the reactive intermediate is able to
inactivate the P450: covalent adduction to an amino acid residue within the
enzyme active site, arylation or alkylation of the prosthetic heme moiety, and
destruction of the heme group, leading to heme-derived products that form
crosslinks with the P450 apoprotein (Osawa
and Pohl, 1989).
The kinetic scheme for mechanism-based inactivation
(Silverman, 1995) is shown in
Scheme 1, where E is defined as
the enzyme and I represents the inactivator. Briefly, the inactivator
reversibly binds to the enzyme active site and is catalytically converted to a
reactive intermediate, I*. The reactive intermediate can either be released as
product (P) or it can become covalently adducted within the active site,
leading to an irreversible inactivation of the enzyme (EI
).
A number of criteria are routinely used to determine whether a substrate for a
particular P450 is a mechanism-based inactivator, including time- and
concentration-dependent enzyme inactivation, an absolute requirement for
NADPH, substrate protection against P450 inactivation, irreversibility of
inactivation by dialysis or gel filtration, and a lack of effect on the rate
of inactivation in the presence of exogenous nucleophiles
(Kent et al., 2001).
In 1995, Dexter and Hager reported for the first time the transient heme
N-alkylation of the enzyme chloroperoxidase (CPO) by terminal alkenes
and alkynes (Dexter and Hager,
1995). These data suggested that chloroperoxidase was inactivated
in a P450-type reaction involving the mechanism-based formation of
N-alkylporphyrins during the oxidation of allylbenzene and 1-hexyne.
These structurally distinct compounds inactivated chloroperoxidase in a time-
and concentration-dependent manner with losses in the enzymatic activity
corresponding to losses in the native heme and the formation of
N-alkyl heme adducts. The inactivated CPO formed in these reactions
then underwent a spontaneous loss of the heme adducts (as observed by
electrospray mass spectrometry analysis) with a restoration of enzymatic
activity and native heme (Dexter and Hager,
1995; Debrunner et al.,
1996). At the time Dexter and Hager
(1995) reported these results,
this type of reversible inactivation mechanism had not been previously
reported for CPO or the cytochrome P450 enzymes.
This review will attempt to summarize recent data obtained on the
reversible inactivation of P450s from the 2E and 2B subfamilies of cytochrome
P450 enzymes by small tert-butyl acetylenic compounds. Interestingly,
the reversibility of P450 inactivation can be influenced by both the
architecture of the enzyme active site and changes in the structure of the
inactivator. It may be that reversibility in P450 systems is more common and,
in fact, more complex, than previously thought. Future experiments with P450s
from other families and subfamilies that specifically probe compounds for a
reversible inactivation mechanism will greatly aid in our understanding of
this interesting phenomenon. The importance of reversibility mechanisms in
vivo must also be assessed and may alter our current interpretation of the
mechanism-based inactivation of human cytochrome P450 enzymes by xenobiotics
and clinically relevant drugs.
|
Acetylenic Mechanism-Based Inactivators
|
|---|
A variety of different compounds containing an acetylenic functional group
have been shown to inactivate P450 enzymes in a mechanism-based manner. Ortiz
de Montellano and coworkers have described two mechanisms for the inactivation
of cytochrome P450 enzymes by acetylenes (Ortiz de Montellano,
1985,
1991;
Ortiz de Montellano and Komives,
1985; Ortiz de Montellano and
Reich, 1986). Insertion of the oxygen atom from the P450-derived
activated oxygen species at the internal carbon of the acetylene results in
the formation of a reactive intermediate that leads to heme adduction and
destruction of the heme chromophore. Transfer of the oxygen atom to the
terminal acetylenic carbon results in a reactive intermediate that undergoes
rearrangement to form a ketene species. This reactive ketene can either be
hydrolyzed to produce a carboxylic acid product or it can acylate nucleophilic
amino acid residues within the active site of P450s, resulting in enzyme
inactivation. Examples of both types of reactions have been reported and are
shown in Fig. 1 for
comparison.
The construction of acetylene-derived mechanism-based inactivators for the
cytochromes P450 has used a variety of different carrier structures, including
fatty acids (Ortiz de Montellano and
Reich, 1984; Shak et al.,
1985; CaJacob and Ortiz de
Montellano, 1986; Muerhoff et
al., 1989), aromatic hydrocarbons
(Gan et al., 1984;
Ortiz de Montellano and Komives,
1985; Komives and Ortiz de
Montellano, 1987; Hammons et
al., 1989), and steroids (Covey
et al., 1981; Nagahisa et al.,
1983; Halpert et al.,
1989). Previously, the inactivation of P450 2B1 by substituted
phenylacetylenes was characterized and shown to primarily result in heme
alkylation (Komives and Ortiz de
Montellano, 1987). Another acetylene, 2-ethynylnaphthalene, has
been shown to inactivate purified rat P450s 1A1 and 1A2 in a mechanism-based
manner through covalent adduction of the apoproteins
(Hammons et al., 1989).
Hollenberg and coworkers demonstrated the mechanism-based inactivation of
P450s 2E1 and 2E1 T303A by two structurally similar compounds
(Fig. 2) that contained a
tert-butyl moiety for P450 2E1 specificity and an ethynyl functional
group for P450-dependent metabolism to a reactive intermediate capable of
covalently modifying the active site through heme or protein adduction
(Blobaum et al., 2002).
tert-Butyl acetylene (tBA) and tert-butyl
1-methyl-2-propynyl ether (tBMP) inactivated the P450s 2E1 through three
distinct mechanisms: 1) covalent alkylation of the heme prosthetic moiety (tBA
and tBMP inactivation of P450s 2E1 and 2E1 T303A), 2) a combination of heme
alkylation and protein adduction (tBA inactivation of P450 2E1), and 3) a
novel and not previously described reversible alkylation of the P450 heme (tBA
inactivation of the T303A mutant). Characterization of this reversible
inactivation mechanism demonstrated that losses in the activity and native
heme of a tBA-inactivated T303A sample could be restored by dialysis or spin
column gel filtration (Blobaum et al.,
2004a). Acetylene heme adducts with m/z of 661
Da were shown to be reversible with time, as described for the reversible
inactivation of chloroperoxidase by allylbenzene
(Dexter and Hager, 1995).
Interestingly, a source of exogenous protons was required to observe stable
heme adducts in the tBA-inactivated T303A mutant, whereas the wild-type P450
2E1 was able to form these tBA adducts under the same conditions regardless of
prior preacidification (Blobaum et al.,
2004a). These data suggested an important role for the highly
conserved threonine 303 residue as a possible participant in a proton relay
network to the active site of P450 2E1. In addition, studies with alternate
oxidants capable of supporting enzyme inactivation in the absence of NADPH and
reductase suggested the formation and utilization of a hydroperoxo-iron
species for substrate oxygenation by the T303A mutant and an iron-oxo species
for use by the wild-type 2E1 enzyme, confirming the disruption of proton
delivery to the active site of the threonine mutant
(Blobaum et al., 2004b).
Scheme 2 depicts a speculative
mechanism for the reversible inactivation of P450 2E1 T303A by a small
acetylene such as tBA. In this chemical scheme, an inactivating intermediate
is formed from the insertion of oxygen into an acetylenic inhibitor (tBA) by a
hydroperoxy-iron species in the T303A mutant
(Blobaum et al., 2004a). This
intermediate is responsible for the reversible loss in the enzymatic activity
of 2E1 T303A and is shown in the scheme to have two possible fates: 1) the
formation of the intermediate is reversible over time and will re-form an
activated enzyme with intact heme and an acetylene-derived carboxylic acid
reversal product; or 2) the inactivating intermediate is stabilized in the
presence of exogenous protons and will lead to irreversible
N-alkylation of the P450 heme. It is this second pathway that is
identical to the sequence of steps that are involved in the irreversible
inactivation of the wild-type P450 2E1 enzyme by tBA.
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 2. Structures of two tert-butyl acetylenic compounds. tBA and tBMP
are two structurally similar acetylenic compounds that contain a
tert-butyl moiety for P450 2E1 specificity and an ethynyl functional
group for P450-dependent metabolism to a reactive intermediate capable of
covalently modifying the active site.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
SCHEME 2. Chemical mechanism for the reversible inactivation of P450 2E1 T303A by
small acetylenes. Under oxidative conditions, the hydroperoxy-iron species in
the T303A mutant inserts an oxygen into the acetylenic compound and forms an
inactivating intermediate (in brackets) that can be observed spectrally at 485
nm. This intermediate is responsible for the observed experimental losses in
enzymatic activity of the 2E1 mutant and can either reverse over time to
regenerate the native heme and one or more reversal products or can
N-alkylate the P450 heme in the presence of exogenous protons and
irreversibly modify the enzyme (Blobaum et
al., 2004b).
|
|
|
Unique Spectral Intermediates Formed during Reversible Inactivation
|
|---|
Clorgyline was previously shown to inactivate P450 2B1 through the
formation of a metabolic intermediate (MI) complex that could be reversed to
regenerate the active enzyme (Sharma et
al., 1996). Incubation with clorgyline and NADPH resulted in an
absorbance peak with a maximum at 455 nm in the difference spectrum that is
characteristic of a MI complex. Although MI complexes have been well
documented in the literature and are known to have an absorbance maximum at
455 nm, there has also been evidence for the formation of other types of
spectral intermediate complexes that are characterized by absorbance maxima at
longer wavelengths (Battioni et al.,
1983; Mansuy and Fontecave,
1983). To investigate the possible formation of a metabolic
intermediate complex or some other type of spectral intermediate by the
tBA-inactivated T303A mutant of P450 2E1, extensive spectral analyses were
performed (Blobaum et al.,
2004b). Interestingly, a peak having an absorption maximum at 485
nm was observed with the 2E1 T303A samples; however, a similar peak was not
detected in the spectrum of the wild-type enzyme. The formation of the 485-nm
peak in the T303A enzyme was monitored with time and showed a rapid
accumulation during the first 10 min with a plateau after 20 min. Importantly,
the formation of the peak at 485 nm required oxygen, and after reversion to
the active enzyme after overnight dialysis, the peak could be regenerated by
the addition of fresh tBA and NADPH
(Blobaum et al., 2004b). Given
that typical MI complexes absorb at 455 nm, this spectral intermediate having
a maximum absorption at 485 nm was believed to be a newly discovered tBA-Fe
intermediate. Because protons are required to support the formation of the
N-alkylated tBA heme adducts
(Blobaum et al., 2004a), this
acetylene-iron spectral intermediate may be a chelated structure in which the
oxygenated acetylene forms a bridge between the heme iron atom and one of the
pyrrole nitrogens of the heme moiety (Fig.
3). Scheme 2
proposes the formation of such an intermediate during the reversible
inactivation of P450 2E1 T303A by tBA. In this scheme, the inactivating
intermediate corresponds to the spectral intermediate observed at 485 nm, and
can either lead to inactivation of the P450 by heme adduction or can slowly
decompose over time to regenerate the native enzyme with full catalytic
activity.
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 3. A hypothetical structure for the novel P450 spectral intermediate with an
absorbance maximum at 485 nm. Metabolite intermediate complexes typically
absorb at 455 nm and consist of an intermediate that is complexed solely to
the heme iron. Upon the addition of an exogenous source of protons in the
T303A mutant enzyme, a stable N-alkylated tBA heme product is formed
and can be detected using liquid chromatography-tandem mass spectrometry
analysis. A chelated intermediate that bridges the heme iron, oxygen, the
remaining acetylene, and the pyrrole nitrogen is a suitable candidate for the
reversible spectral intermediate with a 30-nm shift in absorbance to 485 nm.
The addition of a proton to this intermediate could result in the formation of
a N-alkylated tBA heme product
(Blobaum et al., 2004b).
|
|
|
The Role of P450 Active Site Architecture in Reversibility
|
|---|
Active site models of P450 2E1 have shown a highly conserved threonine
(T303) positioned directly over the plane of the heme moiety and within
hydrogen bonding distance to the activated oxygen species
(Tan et al., 1997). Over the
years, many groups have proposed that this threonine residue (T303 in P450
2E1, T302 in P450 2B4, and T252 in P450cam) is involved in a proton
delivery network that shuttles protons to the activated oxygen species in the
enzyme active site (Imai et al.,
1989; Raag et al.,
1991; Vaz et al.,
1996,
1998;
Jin et al., 2003). Much of our
understanding of the important role played by this conserved threonine residue
in proton relay and dioxygen activation has come from studies with the camphor
monooxygenase, P450cam. Mutagenesis of T252 to aliphatic residues
in P450cam led to a loss in enzymatic activity and the production
of peroxide in what is generally considered to be an uncoupling reaction
(Imai et al., 1989;
Martinis et al., 1989). The
X-ray crystal structure of the T252A mutant in the ferric state indicated a
role for this conserved residue in a proton shuttle pathway that connects the
surface of the protein with the enzyme active site
(Raag et al., 1991). The
elucidation of the ferrous dioxygen complex of P450cam provided
evidence for a continuous hydrogen-bonded link between the conserved T252,
water molecules, and the dioxygen ligand
(Schlichting et al., 2000).
Recently, the T252A ferrous dioxygen structure was solved by Nagano and Poulos
(2005) and was unexpectedly
shown to contain two "catalytic" waters in the active site,
similar to waters that are observed in the ferrous dioxygen complex of
wild-type P450cam. In addition, the replacement of the free
hydroxyl group (OH) on T252 with a methoxy group (OCH3) does not
significantly alter the catalytic activity of the enzyme
(Kimata et al., 1995). It has
thus been suggested that this conserved threonine residue may not serve as a
proton donor in dioxygen activation but, rather, may promote the addition of
the second proton in the P450 catalytic cycle to the distal oxygen by
accepting a hydrogen bond from the hydroperoxy-iron species. Finally, and of
great interest, these results demonstrate that even in the absence of the
conserved threonine, water molecules or other compensatory networks for proton
delivery may form in the enzyme active site. As the crystal structures of the
threonine mutants of mammalian P450s in either the ferric or ferrous dioxygen
states are unavailable at this time, it is not known whether these same
observations will hold true.
Threonine 303 in the mammalian cytochrome P450 2E1 was shown to be
important in the mechanism-based inactivation of P450s 2E1 and 2E1 T303A by
the small tert-butyl acetylene, tBA (Blobaum et al.,
2002,
2004a). In the 2E1 mutant
lacking the conserved threonine, a reversible inactivation mechanism with tBA
and the formation of a novel P450 spectral intermediate was observed
(Blobaum et al., 2004b). Models
of the P450s 2E1 and 2E1 T303A with tBA docked in the active site were
generated based on homology with the known crystal structures of the P450 2C
isozymes (Williams et al.,
2000; Williams et al.,
2003). These models show significant differences in the hydrogen
bonding networks between the wild-type 2E1 and T303A mutant enzymes (D. L.
Harris, personal communication). Wild-type 2E1 has two possible networks for
proton delivery to the active site: one involves the delivery of protons down
a network formed entirely of structured water molecules and the second
consists of proton delivery through ordered water molecules and the conserved
amino acid residues, E302 and T303 (Fig.
4A). In the model of 2E1 T303A with tBA, the first network
composed of structured water molecules is still intact, but the second network
with E302 and T303 is completely absent with the T303A mutation
(Fig. 4B). This finding
suggests that proton delivery with this pathway is not occurring in the T303A
mutant and may help to explain the dependence of protons for stable adduct
formation in the tBA-inactivated T303A enzyme. Indeed, it also points to a
role for T303 in the mechanism of reversibility observed in the 2E1 T303A
mutant.
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 4. A, hydrogen bond connectivity in wild-type P450 2E1 with tBA bound in the
active site. Two hydrogen bond networks terminate on the heme-bound dioxygen
of the reduced oxyferrous species in the model of wild-type 2E1 with tBA. The
first network consists entirely of ordered water molecules that terminate
proton delivery on the peptide itself. Bulk water molecules and Glu302 (E302)
connect to the proximal oxygen via structural waters connecting to Thr303
(T303) in the second hydrogen-bonding network (D. L. Harris, personal
communication). B, hydrogen bond connectivity in mutant P450 2E1 T303A with
tBA bound in the active site. Although waters are present in the binding site,
one loses a network connection to E302 and bulk waters. In this model, A303
neither provides stabilization of the bound dioxygen in the active site nor
orders local waters (D. L. Harris, personal communication).
|
|
Given that threonine 303 is highly conserved among P450 enzymes and is
thought to be involved in proton delivery to the active sites of these P450s,
the role of this conserved residue and proton relay networks in reversibility
was examined in P450 2B4 and its T302A mutant. Research showed that the same
acetylenic inactivators (tBA and tBMP) were able to inactivate the two P450s
in a mechanism-based manner, through the formation of acetylene adducts to the
P450 heme (von Weymarn et al.,
2004; Blobaum et al.,
2005). Interestingly, these inactivations were found to be
partially reversible (20–30%) with dialysis and spin column gel
filtration. Protons were required to form stable tBA or tBMP heme adducts in
both wild-type and mutant 2B4 P450s, demonstrating a significant deviation
from the 2E1 studies mentioned above. Active site models of P450 2B4 and the
T302A mutant based on the 2B4 crystal structure
(Scott et al., 2004) showed
that the T302A mutation does not significantly alter the architecture of the
enzyme active site or the proton delivery networks therein
(Blobaum et al., 2005). As with
P450 2E1, two possible networks for proton delivery exist in the 2B4 P450s.
However, the glutamate (E301) and threonine (T302) network remains intact in
the T302A mutant of 2B4, suggesting that there is still efficient delivery of
protons in this enzyme. Thus, it was deduced from mass spectral data and
computational modeling that the conserved threonine residue in P450 2B4 is not
involved in the delivery of protons to the acetylene reactive intermediate of
the heme or in the observed partial reversibility with the 2B4 enzymes. It can
be concluded from these studies that active site architecture and proton relay
may play a significant role in the determinants of reversibility in these
P450s. Since these glutamate and threonine residues are highly conserved among
all P450s, targeted mutations in other mammalian P450s may prove useful in
determining their role in proton delivery and reversibility.
|
Chemical Determinants of Reversibility
|
|---|
The chemical nature of the inactivator also seems to play a role in
determining reversibility with P450 enzymes. When P450s 2E1 and 2E1 T303A were
inactivated by tBA and tBMP, only the small tBA compound was able to
demonstrate reversibility with the T303A mutant (Blobaum et al.,
2002,
2004a). The larger tBMP
inactivated the 2E1 T303A enzyme in an irreversible manner, and it was thought
that the internal oxygen moiety of tBMP and its elongated structure may have
provided more surface contacts for stabilizing hydrogen bonding in the enzyme
active site. Terminal acetylenes and not their olefin counterparts are capable
of inactivation and reversibility with P450s 2E and 2B (unpublished
observation). For example, in testing the double-bonded tert-butyl
olefin for its ability to reversibly inactivate the 2E1 T303A mutant in a
mechanism-based manner, inactivation of the enzyme was not observed. This
suggested a requirement of an acetylene group for metabolic activation to a
reactive intermediate capable of inactivating the enzyme. The relative size
and chemical nature of the functional groups attached to terminal acetylenic
compounds also influence the determinants of reversibility with P450 2E1 and
the 2E1 T303A mutant. Several additional acetylenes
(Fig. 5) were tested for their
ability to inactivate these P450s in a mechanism-based manner and were
monitored for their ability to reverse these inactivations with dialysis and
spin column gel filtration (Table
1; unpublished observations). Larger, aromatic acetylenes (tBPA)
were only competitive inhibitors, whereas elongated acetylenes without
internal oxygen atoms (3-PP) were irreversible mechanism-based inactivators of
both 2E1 P450s. Small terminal acetylenes (4-MP), on the other hand, were able
to reversibly inactivate the T303A mutant of 2E1 in a mechanism-based manner.
Spectral analyses showed the formation of a P450 spectral intermediate at 483
nm with 4-MP and a requirement for protons to form stable acetylene heme
adducts (Fig. 6).
Interestingly, the structural requirement for reversibility does not seem to
involve the tert-butyl moiety found in tBA, since 4-MP lacks this
functional group. However, there does seem to be an appropriate size
requirement at this location since acetylenes containing larger aromatic
functional groups will only show mechanism-based inactivation or competitive
inhibition, not reversibility.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 5. Structures of three substituted acetylenes: 4-MP, tBPA, and 3-PP. These
three acetylenes were studied in an attempt to probe the structural
determinants for the inactivation and reversibility of inactivation of P450s
2E1 by small acetylenic compounds. The structures include a linear acetylene
(4-MP), a tert-butyl-substituted aromatic acetylene (tBPA), and a
simple aromatic acetylene (3-PP).
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 1 Slight changes in inactivator structure effect the inactivation and the
reversibility of inactivation of P450 2E1 T303A by acetylenes
|
|
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 6. Time-dependent formation of the 4-MP-Fe spectral intermediate at 483 nm.
This figure shows scans of the difference spectrum between the control and
4-MP-inactivated T303A sample taken at 90-s intervals and illustrates the
time-dependent formation of the 483-nm peak.
|
|
|
Conclusions and Future Directions
|
|---|
The topic of reversibility in the context of mechanism-based inactivation
of P450 enzymes has never really been considered an object of much discussion.
It was thought that compounds that fulfilled the criteria of a mechanism-based
inactivator should show an irreversible inactivation mechanism, indicative of
a covalent modification of the enzyme active site resulting in a permanent
loss of enzyme activity. The observation that mechanism-based inactivators can
fulfill all the criteria set forth by Silverman
(1995) and yet can inactivate
in a reversible manner is a new trend in P450 literature. The reversible
inactivations of chloroperoxidase and P450 enzymes by acetylenic compounds are
the first such reports of reversible mechanism-based inactivators. As
discussed in this review, it is apparent that there are multiple determinants
of reversibility. Enzyme active site architecture and the location and
distance of critical amino acids residues from the inactivator itself seem to
influence the mechanism of reversibility. In addition, the size and chemical
nature of the compound will determine whether it is an irreversible or a
reversible mechanism-based inactivator. Is the reversible inactivation of
P450s a new trend that we must consider in terms of our viewpoints on P450
inhibition literature and P450-inactivator relationships? Experiments that
answer these critical questions and determine the enzymatic and chemical
requirements for reversibility are crucial for our understanding of P450
inactivation in biological systems.
View larger version (127K):
[in this window]
[in a new window]
|
Anna Blobaum received her Bachelor of Arts degrees from West
Virginia University in chemistry and biology in 1999. She completed her
dissertation in the department of pharmacology in the laboratory of Dr. Paul
Hollenberg at the University of Michigan in 2004. Her thesis was entitled
"Mechanism-Based Inactivation of Cytochromes P450 2E1 and 2E1 T303A by
tert-Butyl Acetylenes." In 2003 and 2004, Dr. Blobaum was
awarded excellence in teaching and excellence in research/service honors from
the University of Michigan Medical
School.
|
|
|
Acknowledgments
|
|---|
I wish to acknowledge Dr. Paul Hollenberg and Dr. Ute Kent (University of
Michigan) and Dr. William Alworth (Tulane University) for helpful discussions.
I would also like to make a special acknowledgment to Dr. Danni Harris
(Molecular Research Institute) for P450 models generated for panels A and B of
Fig. 4 and for insights into
the proton delivery pathways of P450s.
|
Footnotes
|
|---|
Supported in part by National Institutes of Health Grants CA 16954 (Paul F.
Hollenberg), GM 07767 (Anna L. Blobaum), DA 017029 (Anna L. Blobaum), and
R43-DC-6925 (Danni L. Harris).
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.004747.
ABBREVIATIONS: P450, cytochrome P450; tBA, tert-butyl
acetylene; tBMP, tert-butyl 1-methyl-2-propynyl ether; CPO,
chloroperoxidase; 4-MP, 4-methyl-1-pentyne; tBPA,
4-tert-butylphenylacetylene; 3-PP, 3-phenyl-1-propyne.
Dr. Blobaum is currently a postdoctoral fellow in the laboratory of Dr.
Lawrence J. Marnett in the biochemistry department at Vanderbilt University,
and her work focuses on the determinants of the selectivity for cyclooxygenase
enzyme inhibition by structurally novel COX-2 inhibitors.
Address correspondence to: Dr. Anna L. Blobaum, Department of
Biochemistry, Vanderbilt University, 854 Robinson Research Building,
23rd Avenue South at Pierce, Nashville, TN 37232. E-mail:
anna.l.blobaum{at}vanderbilt.edu
|
References
|
|---|
Battioni P, Mahy JP, Delaforge M, and Mansuy D (1983)
Reaction of monosubstituted hydrazines and diazenes with rat-liver cytochrome
P450. Formation of ferrous-diazene and ferric sigma-alkyl complexes.
Eur J Biochem 134:241
–248.[Medline]
Blobaum AL, Harris DL, and Hollenberg PF (2005) P450
active site architecture and reversibility: inactivation of cytochromes P450
2B4 and 2B4 T302A by tert-butyl acetylenes.
Biochemistry 44:3831
–3844.[CrossRef][Medline]
Blobaum AL, Kent UM, Alworth WL, and Hollenberg PF
(2002) Mechanism-based inactivation of cytochromes P450 2E1 and
2E1 T303A by tert-butyl acetylenes: characterization of reactive intermediate
adducts to the heme and apoprotein. Chem Res Toxicol
15:1561
–1571.[CrossRef][Medline]
Blobaum AL, Kent UM, Alworth WL, and Hollenberg PF
(2004a) Novel reversible inactivation of cytochrome P450 2E1
T303A by tert-butyl acetylene: the role of threonine 303 in proton delivery to
the active site of cytochrome P450 2E1. J Pharmacol Exp
Ther 310:281
–290.[Abstract/Free Full Text]
Blobaum AL, Lu Y, Kent UM, Wang S, and Hollenberg PF
(2004b) Formation of a novel reversible cytochrome P450 spectral
intermediate: role of threonine 303 in P450 2E1 inactivation.
Biochemistry 43:11942
–11952.[Medline]
CaJacob CA and Ortiz de Montellano PR (1986)
Mechanism-based in vivo inactivation of lauric acid hydroxylases.
Biochemistry 25:4705
–4711.[CrossRef][Medline]
Covey DF, Hood WF, and Parikh VD (1981) 10
beta-propynyl-substituted steroids. Mechanism-based enzyme-activated
irreversible inhibitors of estrogen biosynthesis. J Biol
Chem 256:1076
–1079.[Abstract/Free Full Text]
Debrunner PG, Dexter AF, Schulz CE, Xia YM, and Hager LP
(1996) Mossbauer and electron paramagnetic resonance studies of
chloroperoxidase following mechanism-based inactivation with allylbenzene.
Proc Natl Acad Sci USA
93:12791
–12798.[Abstract/Free Full Text]
Dexter AF and Hager LP (1995) Transient heme
N-alkylation of chloroperoxidase by terminal alkenes and alkynes. J
Am Chem Soc 117:817
–818.[CrossRef]
Gan LS, Acebo AL, and Alworth WL (1984)
1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent
benzo[a]pyrene hydroxylase activity in liver microsomes.
Biochemistry 23:3827
–3836.[CrossRef][Medline]
Halpert J, Jaw JY, and Balfour C (1989) Specific
inactivation by 17 beta-substituted steroids of rabbit and rat liver
cytochromes P-450 responsible for progesterone 21-hydroxylation.
Mol Pharmacol 35:148
–156.[Abstract]
Hammons GJ, Alworth WL, Hopkins NE, Guengerich FP, and Kadlubar FF
(1989) 2-Ethynylnaphthalene as a mechanism-based inactivator of
the cytochrome P-450 catalyzed N-oxidation of 2-naphthylamine. Chem
Res Toxicol 2:367
–374.[CrossRef][Medline]
Imai M, Shimada H, Watanabe Y, Matsushima-Hibiya Y, Makino R, Koga
H, Horiuchi T, and Ishimura Y (1989) Uncoupling of the cytochrome
P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine
or valine: possible role of the hydroxy amino acid in oxygen activation.
Proc Natl Acad Sci USA
86:7823
–7827.[Abstract/Free Full Text]
Jin S, Makris TM, Bryson TA, Sligar SG, and Dawson JH
(2003) Epoxidation of olefins by hydroperoxo-ferric cytochrome
P450. J Am Chem Soc 125:3406
–3407.[CrossRef][Medline]
Kent UM, Juschyshyn MI, and Hollenberg PF (2001)
Mechanism-based inactivators as probes of cytochrome P450 structure and
function. Curr Drug Metab
2:215
–243.[CrossRef][Medline]
Kimata Y, Shimada H, Hirose T, and Ishimura Y (1995)
Role of Thr-252 in cytochrome P450cam: a study with unnatural amino acid
mutagenesis. Biochem Biophys Res Commun
208:96
–102.[CrossRef][Medline]
Komives EA and Ortiz de Montellano PR (1987) Mechanism
of oxidation of pi bonds by cytochrome P-450. Electronic requirements of the
transition state in the turnover of phenylacetylenes. J Biol
Chem 262:9793
–9802.[Abstract/Free Full Text]
Mansuy D and Fontecave M (1983) Reduction of benzyl
halides by liver microsomes. Formation of 478 NM-absorbing sigma-alkyl-ferric
cytochrome P-450 complexes. Biochem Pharmacol
32:1871
–1879.[Medline]
Martinis SA, Atkins WM, Stayton PS, and Sligar SG
(1989) A conserved residue of cytochrome P-450 is involved in
heme-oxygen stability and activation. J Am Chem Soc
111:9252
–9253.[CrossRef]
Massey V, Komai H, Palmer G, and Elion GB (1970) On
the mechanism of inactivation of xanthine oxidase by allopurinol and other
pyrazolo[3,4-d]pyrimidines. J Biol Chem
245:2837
–2844.[Abstract/Free Full Text]
Muerhoff AS, Williams DE, Reich NO, CaJacob CA, Ortiz de Montellano
PR, and Masters BS (1989) Prostaglandin and fatty acid omega- and
(omega-1)-oxidation in rabbit lung. Acetylenic fatty acid mechanism-based
inactivators as specific inhibitors. J Biol Chem
264:749
–756.[Abstract/Free Full Text]
Nagahisa A, Spencer RW, and Orme-Johnson WH (1983)
Acetylenic mechanism-based inhibitors of cholesterol side chain cleavage by
cytochrome P-450scc. J Biol Chem
258:6721
–6723.[Abstract/Free Full Text]
Nagano S and Poulos TL (2005) Crystallographic study
on the dioxygen complex of wild-type and mutant cytochrome P450cam:
implications for the dioxygen activation mechanism. J Biol
Chem 280:31659
–31663.[Abstract/Free Full Text]
Ortiz de Montellano PR (1985) Alkenes and alkynes, in
Bioactivation of Foreign Compounds (Anders MW ed) pp121
–155, Academic Press, New York.
Ortiz de Montellano PR (1991) Mechanism-based
inactivation of cytochrome P450: isolation and characterization of N-alkyl
heme adducts. Methods Enzymol
206:533
–540.[Medline]
Ortiz de Montellano PR and Komives EA (1985)
Branchpoint for heme alkylation and metabolite formation in the oxidation of
arylacetylenes by cytochrome P-450. J Biol Chem
260:3330
–3336.[Abstract/Free Full Text]
Ortiz de Montellano PR and Reich NO (1984) Specific
inactivation of hepatic fatty acid hydroxylases by acetylenic fatty acids.
J Biol Chem 259:4136
–4141.[Abstract/Free Full Text]
Ortiz de Montellano PR and Reich NO (1986) Inhibition
of cytochrome P450 enzymes, in Cytochrome P450: Structure,
Mechanism and Biochemistry (Ortiz de Montellano PR ed) pp273
–314, Plenum Press, New York.
Osawa Y and Pohl LR (1989) Covalent bonding of the
prosthetic heme to protein: a potential mechanism for the suicide inactivation
or activation of hemoproteins. Chem Res Toxicol
2:131
–141.[CrossRef][Medline]
Raag R, Martinis SA, Sligar SG, and Poulos TL (1991)
Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala.
Biochemistry 30:11420
–11429.[CrossRef][Medline]
Rando RR (1984) Mechanism-based enzyme inactivators.
Pharmacol Rev 36:111
–142.[Medline]
Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, Benson DE,
Sweet RM, Ringe D, Petsko GA, and Sligar SG (2000) The catalytic
pathway of cytochrome p450cam at atomic resolution. Science (Wash
DC) 287:1615
–1622.[Abstract/Free Full Text]
Scott EE, White MA, He YA, Johnson EF, Stout CD, and Halpert JR
(2004) Structure of mammalian cytochrome P450 2B4 complexed with
4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of
P450 conformations and the coordination of redox partner binding. J
Biol Chem 279:27294
–27301.[Abstract/Free Full Text]
Shak S, Reich NO, Goldstein IM, and Ortiz de Montellano PR
(1985) Leukotriene B4 omega-hydroxylase in human
polymorphonuclear leukocytes. Suicidal inactivation by acetylenic fatty acids.
J Biol Chem 260:13023
–13028.[Abstract/Free Full Text]
Sharma U, Roberts ES, and Hollenberg PF (1996)
Formation of a metabolic intermediate complex of cytochrome P4502B1 by
clorgyline. Drug Metab Dispos
24:1247
–1253.[Abstract]
Silverman RB (1995) Mechanism-based enzyme
inactivators. Methods Enzymol
249:240
–283.[Medline]
Tan Y, White SP, Paranawithana SR, and Yang CS (1997)
A hypothetical model for the active site of human cytochrome P4502E1.
Xenobiotica 27:287
–299.[CrossRef][Medline]
Vaz AD, McGinnity DF, and Coon MJ (1998) Epoxidation
of olefins by cytochrome P450: evidence from site-specific mutagenesis for
hydroperoxo-iron as an electrophilic oxidant. Proc Natl Acad Sci
USA 95:3555
–3560.[Abstract/Free Full Text]
Vaz AD, Pernecky SJ, Raner GM, and Coon MJ (1996)
Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in
cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine
mutagenesis of cytochrome P450 2B4. Proc Natl Acad Sci
USA 93:4644
–4648.[Abstract/Free Full Text]
von Weymarn LB, Blobaum AL, and Hollenberg PF (2004)
The mechanism-based inactivation of p450 2B4 by tert-butyl 1-methyl-2-propynyl
ether: structural determination of the adducts to the p450 heme.
Arch Biochem Biophys
425:95
–105.[CrossRef][Medline]
Williams PA, Cosme J, Sridhar V, Johnson EF, and McRee DE
(2000) Mammalian microsomal cytochrome P450 monooxygenase:
structural adaptations for membrane binding and functional diversity.
Mol Cell 5:121
–131.[CrossRef][Medline]
Williams PA, Cosme J, Ward A, Angove HC, Matak VD, and Jhoti H
(2003) Crystal structure of human cytochrome P450 2C9 with bound
warfarin. Nature (Lond)
424:464
–468.[CrossRef][Medline]
This article has been cited by other articles:
|
|
|
|
 
D. Hallifax and J. B. Houston
Saturable Uptake of Lipophilic Amine Drugs into Isolated Hepatocytes: Mechanisms and Consequences for Quantitative Clearance Prediction
Drug Metab. Dispos.,
August 1, 2007;
35(8):
1325 - 1332.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. W. Locuson and T. S. Tracy
Identification of Binding Sites of Non-I-Helix Water Molecules in Mammalian Cytochromes P450
Drug Metab. Dispos.,
December 1, 2006;
34(12):
1954 - 1957.
[Abstract]
[Full Text]
[PDF]
|
|
|
![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Top
Abstract
Mechanism-Based Inactivation and...
