0090-9556/99/2706-0741–745$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 27, No. 6
Printed in U.S.A.
MECHANISM-BASED INACTIVATION OF RAT LIVER CYTOCHROME P-450 2B1 BY 2METHOXY-5-NITROBENZYL BROMIDE
ARTHUR P. ARMSTRONG
AND
PAUL F. HOLLENBERG
Department of Pathology (A.P.A., P.F.H.), Northwestern University School of Medicine, Chicago, Illinois; and Department of Pharmacology
(P.F.H.), University of Michigan, Ann Arbor, Michigan
(Received July 22, 1998; accepted February 8, 1999)
This paper is available online at http://www.dmd.org
ABSTRACT:
in a time- and NADPH-dependent manner, and the loss of activity
followed pseudo-first-order kinetics. The inactivation also required
KR-II, and the rate of activity loss was dependent on the concentration of KR-II in a saturable fashion. The inactivator concentration required for the half-maximal rate of inactivation (KI) was
approximately 0.1 mM. The inactivation was not prevented by the
addition of the nucleophiles dithiothreitol and glutathione, nor was
it reversed by gel filtration. The present results demonstrate that
KR-II is a mechanism-based inactivator of rat CYP2B1.
In many cases, oxidative metabolism by the cytochromes P-450
(P-450)1 critically determines the balance between detoxification and
the formation of toxic products from xenobiotics (Porter and Coon,
1991). The common catalytic function of these enzymes involves a
two-electron reduction of molecular oxygen to form water and a
reactive oxygen species. Because the insertion of the activated oxygen
into substrate is a common feature of all P-450s, the balance between
metabolic activation and detoxification must depend on how a given
substrate binds to a specific form of P-450, which in turn is determined by the structure of the active site of that P-450. One way to find
which peptide regions of a given form of P-450 define its active site
is through identification of the peptides labeled during the inactivation
of that P-450 by mechanism-based inactivators that bind to the apoprotein.
Mechanism-based inactivation occurs when metabolism of the substrate results in the formation of a highly reactive intermediate, which
reacts with a moiety in the active site before leaving the active site and
covalently modifies that amino acid residue, leading to inactivation of
the enzyme. This phenomenon has been reviewed in detail by Abeles
(1983) and Waley (1980). For several classes of mechanism-based
inactivators of P-450, the general reaction sequences proposed to
explain the modification of the active site and the subsequent activity
loss have been reviewed by Ortiz de Montellano and Correia (1983),
Osawa and Pohl (1989), and Murray and Reidy (1990). Several
classes of compounds that inactivate P-450s have been identified
including alkenes, alkynes, dichloromethylenes, and dihydropyridines
(Ortiz de Montellano et al., 1981; Augusto et al., 1982; Kunze et al.,
1983; Ortiz de Montellano et al., 1983; Halpert et al., 1986; CaJacob
et al., 1988).
An alternative to the previously characterized pathways for the
formation of a reactive intermediate leading to active site labeling
involves starting with 2-methoxy-5-nitrobenzyl bromide (Koshland’s
Reagent II, KR-II), which would be expected to undergo P-450catalyzed O-demethylation. The desmethyl analog of KR-II, 2-hydroxy-5-nitrobenzyl bromide, is Koshland’s Reagent I (KR-I). KR-I is
a highly reactive, electrophilic reagent that has been used to label
tryptophan residues (Barman and Koshland, 1967; Loudon and Koshland, 1970). As noted by Horton and Koshland (1967), alkylation of
the phenolic group of KR-I greatly reduces the susceptibility of the
benzyl bromide to nucleophilic attack. Therefore, the resultant esters
and ethers of this phenolic hydroxyl group can serve as precursors of
reactive intermediates. KR-II and KR-I are analogs of the P-450
substrate p-nitroanisole and p-nitrophenol, the product formed during
P-450-catalyzed O-demethylation of p-nitroanisole. Metabolism of
KR-II might occur by several alternate routes, including hydroxylation of the aromatic ring or the benzylic carbon. Conversion of KR-II
to a phenol by either P-450-catalyzed O-demethylation or ring hydroxylation would generate an electrophile at the enzyme active site,
where it could then react with active site nucleophiles. Generation of
an electrophile would be expected to produce mechanism-based inactivation of the enzyme if there were one or more critical nucleophilic residues in the active site with which it could react. Because we
are interested in identifying active site peptides rather than heme
adducts, it was hoped that the electrophilic carbon thus generated
This research was supported in part by National Institutes of Health Grant
CA16954.
1
Abbreviations used are: P-450, cytochrome P-450; CYP2B1, the major form
of cytochrome P-450 from phenobarbital-pretreated rats; KR-I, Koshland’s Reagent I; KR-II, Koshland’s Reagent II; DLPC, dilaurylphosphatidyl choline.
Send reprint requests to: Dr. Paul F. Hollenberg, Department of Pharmacology, Medical Science Research Building III, 1150 West Medical Center Dr., Ann
Arbor, MI 48109-0632. E-mail: phollen@umich. edu
741
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Mechanism-based inactivators serve as probes of enzyme mechanism, function, and structure. Koshland’s Reagent II (2-methoxy5-nitrobenzyl bromide, KR-II) is a potential mechanism-based inactivator of enzymes that perform O-dealkylations. The major
phenobarbital-inducible form of cytochrome P-450 in male rat liver
microsomes, CYP2B1, is capable of catalyzing O-dealkylations.
The interactions of KR-II with purified CYP2B1 in the reconstituted
system containing P-450, NADPH:P-450 oxidoreductase, and sonicated dilaurylphosphatidyl choline were studied. The benzphetamine N-demethylase activity of CYP2B1 was inactivated by KR-II
742
ARMSTRONG AND HOLLENBERG
would not label the heme. Investigation of the enzyme activity levels
before and after incubation of P-450 with KR-II demonstrated that
KR-II acted as a mechanism-based inactivator of P-450 in liver
microsomes from phenobarbital-induced rats and in the reconstituted
system containing CYP2B1.
Materials and Methods
Results
Kinetic and Mechanistic Aspects of Inactivation. To gain a better
understanding of the active site structure and the mechanism of action
of the P-450s, we investigated the inactivation of rat CYP2B1 by
KR-II. Horton and Koshland (1967) developed a clever approach for
modifying hydrolytic enzymes such as the serine proteases that catalyze esterolytic reactions. They discovered that acetylation of the
phenolic hydroxyl group of 2-hydroxy-5-nitrobenzyl bromide results
in a marked decrease in the reactivity of the benzyl bromide function.
Subsequently, it was demonstrated that methylation of the phenolic
hydroxyl to give the methoxy derivative greatly reduced the chemical
reactivity of the benzyl bromide when compared with the unsubstituted form (Lundblad and Noyes, 1984). The major reactions predicted for the P-450-catalyzed metabolism of the methoxy derivative
KR-II, either O-demethylation or ring hydroxylation, would both be
expected to regenerate the reactivity of the benzyl bromide. By
analogy with p-nitroanisole, a well characterized P-450 substrate that
is structurally similar to KR-II, the demethylation route would be
expected to predominate (Fig. 1). This route of metabolism would
generate 2-hydroxy-5-nitrobenzyl bromide, also known as Koshland’s
Reagent I. Koshland’s Reagent I has been used to selectively label
tryptophan residues at low pH (4.0), but at neutral pH it will react with
other nucleophiles in proteins (tyrosine, cysteine, or methionine).
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Preparation of Enzymes. The major phenobarbital-inducible form of liver
microsomal cytochrome P-450 (CYP2B1) was purified from liver microsomes
of male Long Evans rats obtained from Harlan-Sprague-Dawley (Indianapolis,
IN). Microsomes were prepared from the livers of rats induced with 0.1%
phenobarbital in the drinking water for 10 to 13 days as described by Coon et
al. (1978). The CYP2B1 was purified from liver microsomes using the procedure of Imai et al. (1980). The purified P-450 gave a single band on
SDS-polyacrylamide gel electrophoresis and was electrophoretically pure.
Cytochrome P-450 concentrations were determined from the reduced CO
difference spectra by the method of Omura and Sato (1964a,b), using a
micromolar extinction coefficient of 0.091. The specific contents were 11 to 16
nmol/mg protein.
Rat and rabbit NADPH/cytochrome-P-450 oxidoreductases (reductase)
were purified from liver microsomes of phenobarbital-treated male Long
Evans rats and male New Zealand White rabbits (Langshaw, Augusta, MI) by
the single-column procedure of Shephard et al. (1963) using an adenosine
29,-59-diphosphate agarose (ADP-agarose, Sigma A3515; Sigma Chemical
Co., St. Louis, MO ) affinity column. Rat and rabbit reductases functioned
equally well in the reconstituted system with rat CYP2B1. Flavin mononucleotide (1.0 mM) was added to the flavoprotein preparations to ensure full
reconstitution of the reductase protein with the flavin. The protein was concentrated to at least 10 nmol/ml in an Amicon (Danvers, MA) stirred-cell
concentrator and then dialyzed against 50 mM potassium phosphate buffer, pH
7.7, containing 0.1% EDTA and 20% glycerol. The purified reductase proteins
were electrophoretically pure, although some preparations had up to 10% of
the short reductase lacking the N-terminal membrane insertion segment. The
reductase preparations had specific activities in the cytochrome c reductase
assay (Phillips and Langdon, 1962) of 3.3 to 4.6 U/nmol and specific contents
of 10 to 11 nmol/mg determined using an extinction coefficient of 0.0214
mM21 cm21 at 456 nm (Oprian and Coon, 1982). Reductase was stored at
270°C.
Protein Determinations. Protein concentrations were determined by the
method of Lowry et al. (1951) with the following modifications. For purified
proteins in the absence of detergents, assay volumes were reduced 5-fold.
Otherwise, the precipitating modification of Bensadoun and Weinstein (1976),
with 5- or 10-fold reduced volumes, was used. BSA was used for standard
curves.
General Reconstitution Protocol. Purified CYP2B1 and reductase proteins
(3–10 nmol/ml) were incubated together in a test tube for 3 min at 37°C, 5 min
at 30°C, or for more than 30 min on ice before the addition of dilaurylphosphatidyl choline (DLPC) to give 20 to 25 mg/ml in the reaction mixtures. All
three reconstitution protocols gave activities that were the same within 10%.
After addition of DLPC to the reconstitution mixtures, aliquots containing 0.1
to 1.0 nmol of P-450 were added to the primary reaction mixtures, typically in
a final reaction volume of 1 ml. Primary reactions contained CYP2B1, reductase, DLPC, and various concentrations of KR-II (10 mM to 1 mM). These
primary reaction mixtures were preincubated for at least 3 min before initiation
of the reaction by addition of NADPH (0.1–1.0 mmol/ml). Incubations were
performed in 10 to 100 mM potassium phosphate buffer, pH 7.4. The loss of
activity at various times following the addition of NADPH was determined by
measuring benzphetamine N-demethylase activity in a secondary reaction
mixture as described below.
Benzphetamine N-Demethylase Activity. The reconstituted P-450 system
(0.1 nmol of CYP2B1, 0.12 of nmol reductase, and 25 mg of DLPC) was
incubated in 10 mM potassium phosphate buffer (1 ml, pH 7.4) for 10 min at
37°C with 1 mmol of NADPH and 1 mmol of benzphetamine. Both NADPH
and benzphetamine were added from aqueous stock solutions (10 or 100 mM)
in appropriate volumes (100 or 10 ml), respectively. When benzphetamine was
used as the P-450 substrate, reactions were initiated by the final addition of
NADPH; however, when benzphetamine metabolism was used to assess P-450
activity remaining in aliquots taken from prior incubations, an alternate initiation procedure was used. In this case, the aliquot of enzyme from the primary
incubation mixture contained NADPH so that the metabolism of benzphetamine was initiated by the addition of the aliquot. For these incubations,
supplemental amounts of NADPH and DLPC were added to the mixture of
buffer and benzphetamine before the preincubation to achieve final concentrations of 1.0 mmol/ml and 25 mg/ml, respectively. Reactions were terminated
after a 10-min incubation by addition of 500 ml of 60% (w/v) trichloroacetic
acid to give a final concentration of 20% trichloroacetic acid. Controls were
terminated at time zero. The demethylation reaction was linear for more than
15 min. Samples were analyzed for formaldehyde using a modification of the
Nash procedure (1953) as described by Kedderis et al. (1980), except that the
sample absorbance was measured at 412 nm (Pandey et al., 1989). The amount
of formaldehyde formed was determined from the difference between the
absorbance of the incubation mixture and control divided by the slope of the
line for a set of formaldehyde standards (0 –50 or 0 –100 nmol). Turnover
numbers were calculated by dividing the nanomoles of product (formaldehyde)
formed per minute by the nanomoles of P-450 present in the reaction mixture
and are expressed per minute.
Gel Filtration of the P-450 Reconstituted System after Incubation with
Koshland’s Reagent II. Following incubation, the reconstituted system reaction mixtures (1.25–1.60 ml) were chromatographed on 5-ml bed volume
Excellulose GF-5 desalting columns (Pierce, Rockford, IL) to separate the
enzymes from the reactants and products. Columns were initially equilibrated
and then eluted with 100 mM potassium phosphate buffer, pH 7.4. For these
studies, the reaction mixtures consisted of the standard reconstituted system
(1.0 nmol of P-450/ml, 1.2 nmol of reductase/ml, 25 mg of DLPC/ml) plus
KR-II (100 mM) and either NADPH (100 mM stock in water) or water (20 ml)
in a final volume of 2.0 ml of 100 mM potassium phosphate buffer, pH 7.4.
After incubation for 10 min at 37°C, 0.1-ml aliquots were removed and
assayed for benzphetamine N-demethylation activity, and 1.25-ml aliquots of
the primary reaction mixtures were subjected to gel filtration using 100 mM
potassium phosphate buffer to elute the protein fraction. Aliquots (0.2 ml) of
the protein fractions were assayed for benzphetamine N-demethylase activity.
General Reagents. 2-Methoxy-5-nitrobenzyl bromide was purchased from
Aldrich (Milwaukee, WI). Acetone and methanol were Burdick & Jackson
brand HPLC solvents purchased front Baxter Scientific (McGaw Park, IL).
Dithiothreitol, glutathione, NADPH type III, and DLPC were bought from
Sigma Chemical Co. Benzphetamine hydrochloride was supplied by the Upjohn Company (Kalamazoo, MI). All other chemicals used for these studies
were reagent grade or better and were purchased from commercial suppliers.
743
INACTIVATION OF CYP2B1 BY 2-METHOXY-5-NITROBENZYL BROMIDE
TABLE 1
Requirement for both NADPH and Koshland’s Reagent II for inactivation of rat
CYP2B1
The P-450 reconstituted system containing CYP2B1 and reductase was incubated for 10
min at 37°C with the compounds indicated or appropriate solvents as described in Materials
and Methods. Aliquots were taken, and the activity was determined in the benzphetamine Ndemethylase assay.
Addition to the
Primary Reaction Mixture
None
NADPH (1.0 mM)
KR-II (0.1 mM)
KR-II (0.1 mM)
1
NADPH (1.0 mM)
a
Turnover Number
Activity
Remaining
min21
%
65 6 1a
51 6 1
59 6 2
100
78
90
22 6 4
33
Mean 6 S.D., n 5 3.
FIG. 1. Oxidation of Koshland’s Reagent II by P-450.
Reaction with one of these nucleophilic amino acid residues in the
P-450 active site could lead to inactivation of the enzyme. Figure 1
also shows that ring hydroxylation might possibly occur at any of the
three ring hydrogens, leading to the subsequent reaction of the activated benzyl bromide with a protein nucleophile. Based on the chemical reactivity of the ring carbon atoms, the most likely site for ring
hydroxylation would be at the 3 position adjacent to the methoxy
substituent.
Requirement for NADPH for Inactivation of P-450 by Koshland’s Reagent II. P-450-catalyzed metabolism of substrates normally requires NADPH and molecular oxygen. Because mechanismbased inactivation requires the initial metabolism of the inactivator, a
mechanism-based inactivation would be expected to exhibit the same
cofactor requirements as seen for the metabolism of normal substrates.
Therefore, the requirement for NADPH during the inactivation of
CYP2B1 by KR-II was investigated (Table 1). Incubation with methanol, the solvent for KR-II, resulted in no significant loss of activity.
The slight decrease in activity (5–10%) seen with KR-II in the
absence of metabolism was independent of incubation time and seems
to be due to competitive inhibition by KR-II carried over into the
benzphetamine assay with the enzyme. The activity loss (22%) observed following incubation with NADPH in the absence of KR-II is
due to the auto-inactivation of CYP2B1 in the reconstituted system,
which has previously been described (Loosemore et al., 1981). In the
presence of both KR-II and NADPH, however, significant (67%)
inactivation was observed (Table 1).
Dependence on Koshland’s Reagent II Concentration for P-450
Inactivation. Because mechanism-based inactivation is an enzymecatalyzed phenomenon, it would be expected to display a concentration dependence for inactivation similar to that observed for the
metabolism of normal substrates. Thus, the rate of activity loss should
parallel the rate of substrate metabolism. The initial rates for substrate
metabolism normally exhibit saturation kinetics with respect to substrate concentration, as demonstrated by a hyperbolic approach of the
initial rate of the reaction to a limiting maximum rate at saturating
substrate concentrations. The inactivation of CYP2B1 by KR-II was
determined at different concentrations of KR-II to see whether this
pattern was observed. As shown in Fig. 2, the inactivation of CYP2B1
by KR-II exhibited the expected approach to saturation kinetics. The
data are plotted as percent activity lost after a 10-min incubation for
FIG. 2. Concentration dependence for the inactivation of CYP2B1 by Koshland’s
Reagent II.
The P-450 reconstituted system containing CYP2B1 and reductase was incubated
for 10 min at 37°C with the indicated concentrations of KR-II or methanol as
described in Materials and Methods. Aliquots were then taken and diluted to assay
for loss of activity in the benzphetamine N-demethylase assay. The data points
represent the mean 6 S.D., n 5 3.
a range of concentrations of KR-II. From these data, a KI value of 0.1
mM was estimated for KR-II for the mechanism-based inactivation of
CYP2B1.
Protection of CYP2B1 Against Mechanism-Based Inactivation
by Addition of Nucleophiles. For mechanism-based inactivation, the
inactivating intermediate must be generated in the active site and react
there without leaving the active site and diffusing back in or inactivating by reacting elsewhere on the protein. Because mechanismbased inactivators generated by cytochrome P-450 would be expected
to be highly electrophilic, a strong nucleophile added to the reaction
medium should serve as a trapping reagent to prevent back diffusion
of any electrophile(s) escaping into the incubation medium from the
enzyme active site. Thus, the rate of inactivation would be decreased
in the presence of such a trapping agent if the reactive intermediate
responsible for the inactivation were leaving the active site before
inactivating the enzyme.
Because neither dithiothreitol nor glutathione exhibited any significant effects on the rate of CYP2B1-catalyzed benzphetamine Ndemethylation when added to the incubation mixtures at 1 mM concentrations (data not shown), these strong nucleophiles were
investigated as potential trapping agents for reactive electrophiles
released following metabolism at the P-450 active site. As shown in
Table 2, mechanism-based inactivation by KR-II resulted in a 70%
loss of the benzphetamine N-demethylase activity. However neither
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P-450-mediated oxidation of KR-II can result in O-demethylation (1) or ring
hydroxylation (3) to form a nitrophenol. Either product can be attacked by a
nucleophile (2) at its activated benzylic carbon with the nucleophile replacing the
bromine atom.
744
ARMSTRONG AND HOLLENBERG
TABLE 2
TABLE 3
Effects of added nucleophiles on Koshland’s Reagent II inactivation of CYP2B1
Inability to reverse Koshland’s Reagent II inactivation of CYP2B1 by gel
filtration
The P-450 reconstituted system containing CYP2B1 and reductase was incubated for 10
min at 37°C with NADPH (1 mM), KR-II (0.1 mM), and the compounds indicated or their
solvent (water) as described in Materials and Methods. At the end of the incubation, aliquots
were taken and assayed for benzphetamine N-demethylase activity.
Time 0 Control
Water
Glutathione
Dithiothreitol
a
Concentration
Turnover Number
Activity
Remaining
mM
min21
%
1.00
1.00
55 6 1
17 6 6
19 6 2
14 6 9
a
The P-450 reconstituted system containing CYP2B1 and reductase was incubated for 10
min at 37°C with KR-II (50 mM) and either NADPH (1 mM) or water as described in
Materials and Methods. Aliquots were then taken either for immediate analysis or for gel
filtration with subsequent analysis of aliquots for activity in the benzphetamine Ndemethylase assay.
Addition to the Primary
Reaction Mixtures
100
30
34
25
Before gel filtration
Water
NADPH
After gel filtration
Water
NADPH
Mean 6 S.D., n 5 3.
a
Activity
Remaining
min21
%
54 6 1a
37 6 3
100
68
50 6 8
34 6 4
92
62
Mean 6 S.D., n 5 3.
FIG. 3. Loss of activity during P-450-catalyzed metabolism of Koshland’s
Reagent II.
The loss of rat CYP2B1 activity in the reconstituted system containing CYP2B1
and reductase during KR-II (0.10 mM) metabolism was determined by measuring
residual benzphetamine N-demethylase activity. Following the protocol outlined in
Materials and Methods, aliquots (100 ml) of the KR-II metabolism mixture containing 0.1 nmol of rat CYP2B1 and 0.12 nmol of reductase were removed at the
indicated times. The benzphetamine assay mixture (1 ml) contained these proteins
along with DLPC (25 mg), benzphetamine (1 mmol), and NADPH (1 mmol). The
assays were conducted as described in Materials and Methods. The data points
represent the mean 6 S.D., n 5 3.
Discussion
Although benzphetamine N-demethylase activity is expressed by
several different isoforms of P-450, it is a good measure of CYP2B1
activity in the reconstituted system and in liver microsomes. KR-II
was examined for its ability to inactivate benzphetamine N-demethylase activity in liver microsomes from phenobarbital-pretreated rats
and then further tested to see whether the loss of activity was time
dependent. Because KR-II produced a time-dependent inactivation of
P-450 activity in those preliminary studies (data not shown), the
inactivation was subsequently characterized in the reconstituted system containing CYP2B1, reductase, and DLPC to see whether it was
a mechanism-based inactivator.
Loss of CYP2B1 activity in the reconstituted system required the
addition of NADPH, as had been observed in microsomes. The values
for the percentage of activity remaining were determined at various
concentrations of KR-II and were plotted versus the concentration to
see whether the inactivation exhibited saturation kinetics. The concentration dependence for the inactivation of rat CYP2B1 by KR-II
showed, as does normal substrate metabolism, a hyperbolic approach
to a limiting maximum rate. More rigorous methods of kinetic anal-
ysis such as those of Waley (1980, 1985) or Tatsunami et al. (1981)
were not used because this treatment adequately demonstrated the
nature of the inactivation. Loss of CYP2B1 activity during the metabolism of KR-II was first-order with respect to time. The absence of
a lag period in the inactivation kinetics showed that inactivation was
an immediate result of the primary metabolism of KR-II by CYP2B1.
The absence of protection by the exogenous nucleophiles glutathione
and dithiothreitol reinforced the conclusion that this was mechanismbased inactivation. Thus, the inactivation took place when the reactive
intermediate was generated in the active site and before the intermediate could diffuse out of the active site. The inability to reverse the
loss of activity by gel filtration showed the permanence of the inactivating modification, presumably the result of the formation of a
covalent adduct. The characteristics of the inactivation, especially the
pseudo-first-order nature of the KR-II inactivation kinetics, suggest
that inactivation by KR-II fits a simple model where metabolism leads
either to free products or an inactivating modification of the CYP2B1.
Selectivity for protein rather than heme modification during the
mechanism-based inactivation of P-450s has been observed with
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dithiothreitol nor glutathione showed any protection of the enzyme
against KR-II inactivation.
Failure to Reverse Koshland’s Reagent II Inactivation of
CYP2B1 by Gel Filtration. Mechanism-based inactivation involves
covalent modification(s) of the enzyme. It can be differentiated from
product inhibition, which can exhibit many of the same properties, by
separating the enzyme from the substrate and products of the reaction
and assaying for catalytic activity. If product inhibition is involved,
enzymatic activity should be recovered, whereas with mechanismbased inactivation, the loss of enzymatic activity would be irreversible. Gel filtration of the primary incubation mixtures was used to
separate the reconstituted enzymes from the other reactants and products before testing for enzymatic activity. The data shown in Table 3
demonstrate that CYP2B1 inactivated by KR-II did not recover any
activity after separation from the substrate and metabolites. The ratio
of activity in the preparation incubated with NADPH to that of the
preparation incubated with water is the same (0.65) both before and
after gel filtration. Thus, as expected for mechanism-based inactivation, the loss of activity could not be reversed by gel filtration.
Kinetics of CYP2B1 Inactivation by Koshland’s Reagent II.
Enzyme inactivation by mechanism-based inactivators should exhibit
pseudo-first-order kinetics with respect to the enzyme concentration
(Abeles, 1983). As shown in Fig. 3, the inactivation of CYP2B1 by
KR-II exhibited a first-order loss of activity when plotted as the log of
the percentage of activity remaining versus time, consistent with the
hypothesis that KR-II inactivation of CYP2B1 is a mechanism-based
inactivation.
Turnover
Number
INACTIVATION OF CYP2B1 BY 2-METHOXY-5-NITROBENZYL BROMIDE
Acknowledgment. We thank David Putt for assistance in protein
purifications.
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some, but not all, mechanism-based inactivators. Selectivity is dependent in part upon the position of the activatable group when bound in
the active site. For example, Ortiz de Montellano and Komives (1985,
1987) proposed that the regiospecificity of alkyne oxidation determines whether the inactivating intermediate reacts with the heme or
protein. They observed that oxidation of internal carbons leads to
heme alkylation and destroys the P-450 spectrum, whereas oxidation
of terminal carbons produces an intermediate that, after rearrangement
to a ketene, can react with protein nucleophiles or be hydrolyzed to
carboxylic acid products. Consistent with this, CaJacob et al. (1988)
demonstrated that mechanism-based inactivation of P-450 4A1 (the
laurate v hydroxylase) by 10-undecynoic acid results in covalent
modification of the P-450 protein by a ketene intermediate. More
recently, Hopkins et al. (1992) looked at the structure-activity relationships for mechanism-based inactivation of either ethoxyresorufin
O-deethylase (1A1) or pentoxyresorufin O-depentylase (2B1) activity
in microsomes by aryl acetylenes. They found that the position of
attachment of the acetylenic function, along with the size and shape of
the polycyclic aromatic ring system, were all critically important in
determining the selectivity and efficacy of the aryl acetylenes for the
mechanism-based inactivation of these P-450 activities. Roberts et al.
(1993, 1994, 1995) demonstrated that protein labeling occurs during
mechanism-based inactivation of CYP2B1 by 2-ethynylnaphthalene.
The activity loss observed is independent of heme destruction and is
due to modification of the protein with an approximate stoichiometry
of 1.3 mol of [3H]2-ethynylnaphthalene incorporated per mole of
P-450 inactivated. Although the labeled residue has not yet been
unequivocally identified, it is within an 11-amino acid segment of the
CYP2B1 sequence from Phe-297 to Leu-307 (FAGTETSSTTL)
(Roberts et al., 1995). The role of Thr-302 in the mechanism-based
inactivation of CYP2B4 by 2-ethynylnaphthalene has been demonstrated by site-specific mutagenesis (Roberts et al., 1996). HPLC
analysis of CYP2B1 inactivated using 14C-labeled KR-II demonstrated that all of the counts were associated with the apoprotein and
that there were no counts associated with the heme peak (data not
shown). These results suggest that the inactivation of CYP2B1 by
KR-II was due to protein modification rather than heme modification.
Having shown here that KR-II acts as a mechanism-based inactivator of CYP2B1, we are further characterizing this inactivation. The
mechanism(s) involved in inactivation are being investigated by isolating and identifying the metabolites produced during the metabolism
of KR-II by P-450. The studies described here demonstrate an alternative strategy for the design of a mechanism-based inactivator of
P-450. It is hoped that these studies will not only contribute a useful
new mechanistic tool but also aid in the identification of specific
portions of the P-450 protein that participate in the formation of the
active site.
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