Glucuronidation of dihydrotestosterone and trans-androsterone by recombinant UDP-glucuronosyltransferase (UGT) 1A4: Evidence for multiple UGT1A4 aglycone binding sites

Article abstract of DOI:10.1124/dmd.109.028712

UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systema

Full text of DOI:10.1124/dmd.109.028712

0090-9556/10/3803-431–440$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
DMD 38:431–440, 2010
Vol. 38, No. 3
28712/3560030
Printed in U.S.A.
Glucuronidation of Dihydrotestosterone and trans-Androsterone
by Recombinant UDP-Glucuronosyltransferase (UGT) 1A4:
Evidence for Multiple UGT1A4 Aglycone Binding Sites
Jin Zhou, Timothy S. Tracy, and Rory P. Remmel
Departments of Medicinal Chemistry (J.Z., R.P.R.) and Experimental and Clinical Pharmacology (T.S.T.), University of
Minnesota, Minneapolis, Minnesota
Received June 12, 2009; accepted December 3, 2009
ABSTRACT:
UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an inhibited both processes. The glucuronidation kinetics of TAM were then
important drug elimination pathway. Although atypical kinetic profiles evaluated both alone and in the presence of different concentrations of
(nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucu-
DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting
ronidation have been reported occasionally, systematic kinetic studies that TAM may have two binding sites in UGT1A4. However, the substrate
to explore the existence of multiple aglycone binding sites in UGT1A4 inhibition kinetic profile of TAM became more hyperbolic as the DHT or
have not been conducted. To this end, two positional isomers, dihy-
drotestosterone (DHT) and trans-androsterone (t-AND), were used as adequately explained the interactions between TAM and DHT or TAM
probe substrates, and their glucuronidation kinetics with HEK293- and t-AND. In addition, the effect of TAM on LTG glucuronidation was
t-AND concentration was increased. Various two-site kinetic models
expressed UGT1A4 were evaluated both alone and in the presence of a evaluated. In contrast to the mixed effect of TAM on DHT and t-AND
UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest
with TAM, a high-affinity UGT1A4 substrate, resulted in a concentration- that multiple aglycone binding sites exist within UGT1A4, which may
dependent activation/inhibition effect on DHT and t-AND glucuronida- result in atypical kinetics (both homotropic and heterotropic) in a sub-
tion, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively strate-dependent fashion.
Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), domain of human UGT2B7 has recently been published (Miley et al.,
is an important elimination pathway of various endogenous compounds
such as steroid hormones, bile acids, and bilirubin, as well as a large
number of xenobiotics including drugs and their metabolites (Tukey and
Strassburg, 2000). Of the 21 functional human UGT isoforms that have
been characterized to date (Mackenzie et al., 2008), human UGT1A4 is
often considered as the primary catalyst for N-glucuronidation because of
its efficiency in catalyzing the glucuronidation of primary, secondary,
tertiary, and aromatic amines (Kiang et al., 2005). In addition to different
amines, steroidal compounds with hydroxyl groups such as diosgenin and
hecogenin are also UGT1A4 substrates (Green and Tephly, 1996).
Human UGTs are integral membrane proteins, with the majority of
the protein, including the substrate binding sites (both aglycone and
UDPGA), on the luminal side of the endoplasmic reticulum mem-
brane (Radominska-Pandya et al., 1999). Although an apo crystal
structure of the cofactor UDP-glucuronic acid (UDPGA) binding
2007), the three-dimensional structures of the aglycone binding sites
of UGTs are unknown, and the interactions between their aglycone
substrates and the substrate binding sites are poorly understood.
Similar to the cytochromes P450 such as CYP3A4, some UGT iso-
forms also exhibit atypical (non-Michaelis-Menten) kinetic features
(Fisher et al., 2000; Uchaipichat et al., 2004; Iwuchukwu and Nagar,
2008; Ohno et al., 2008). Although the molecular mechanism(s) of
atypical kinetics is still not fully established, numerous studies with the
cytochromes P450 support the hypothesis that simultaneous binding of
multiple molecules to the enzyme is involved (Shou et al., 1994; Kor-
zekwa et al., 1998; Kenworthy et al., 2001; Shou et al., 2001; Galetin et
al., 2002). Such detailed studies with UGTs are less prevalent. Uchaipi-
chat et al. (2008) recently examined 4-methylumbelliferone, 1-naphthol,
and zidovudine glucuronidation by UGT2B7. These authors concluded
that the kinetic data provided evidence for the existence of multiple
aglycone binding sites in UGT2B7. Rios and Tephly (2002) also pro-
posed that two or more aglycone binding sites may exist within UGT1A1,
based on evaluations of the interactions of UGT1A1-catalyzed buprenor-
phine and bilirubin glucuronidation.
This work was supported in part by the National Institutes of Health National
Institute of General Medical Sciences [Grant GM063215] (to T.S.T.); Bristol-Myers
Squibb (to R.P.R.); and a grant from Shimadzu was awarded for the purchase of
the Shimadzu LCMS-2010A instrument used in this study.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
Atypical kinetics of UGT1A4-catalyzed glucuronidation have also
been reported (Chouinard et al., 2006; Hashizume et al., 2008; Hyland
et al., 2009). However, systematic kinetic studies to explore the
doi:10.1124/dmd.109.028712.
ABBREVIATIONS: UGT, UDP glucuronosyltransferase; UDPGA, UDP-glucuronic acid; DHT, dihydrotestosterone; t-AND, trans-androsterone,
epiandrosterone; TAM, tamoxifen; LTG, lamotrigine; HPLC, high-performance liquid chromatography; ESI, electrospray ionization; MS, mass
spectrometry; DMSO, dimethyl sulfoxide; LC, liquid chromatography; RF, radiofrequency; AICc, second-order Akaike information criterion.
431

432
ZHOU ET AL.
bromo-␣-D-glucuronic acid methyl ester were purchased from Sigma-Aldrich
(St. Louis, MO). MgCl2 was purchased from Mallinckrodt (Hazelwood, MO).
All other chemicals used in the glucuronidation incubations, as well as the
HPLC solvents were of HPLC grade. Chemicals used in the synthesis of
tamoxifen-N-glucuronide were ACS grade. Recombinant UGT1A4 was pro-
duced in HEK293 cells (gift from Dr. Philip Lazarus, Penn State University,
Hershey, PA). Cell lysate, prepared by sonication of UGT1A4-HEK293 cells
in 10 mM Tris buffer (pH 7.4 at 37°C) containing 0.25 M sucrose for three 30-s
bursts, each separated by a 1-min cooling on ice, was added directly to the
incubation as the enzyme source. The protein concentration in cell lysate was
determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific,
Waltham, MA).
Synthesis of Tamoxifen-N-glucuronide. Tamoxifen-N-glucuronide was
synthesized according to the method of Kaku et al., 2004. Fifty milligrams
(0.134 mmol) of tamoxifen and 80.2 mg (0.202 mmol) of acetobromo-␣-D-
glucuronic acid methyl ester were dissolved in 0.4 ml of anhydrous dichlo-
romethane and stirred for 72 h at room temperature under nitrogen protection.
The organic solvent was then removed by rotary evaporation. The resulting
residue was dissolved in 3 ml of methanol, and 1.5 ml of 0.5 M aqueous
sodium carbonate was added to the methanolic solution. The resulting solution
was stirred at room temperature for 5 h, and 25 ml of water was then added to
the reaction mixture, which was extracted five times with equal volumes of
ether to remove unreacted tamoxifen. The pH of the aqueous layer was
adjusted to 5.0 with 1 M HCl. Water in the aqueous layer was then removed
by lyophilization. The resulting residue was redissolved with a small volume
of 0.1% formic acid in MeOH and loaded onto a preparative HPLC column
(Haisil HL C18 5 ␮m, 100 ϫ 20 mm; Higgins Analytical Inc., Mountain View,
CA). The tamoxifen glucuronide was eluted with a mobile phase, consisting of
0.1% formic acid in water-0.1% formic acid in MeOH (4:6, v/v), at a flow rate
of 22 ml/min and monitored by UV absorbance at 254 nm. The tamoxifen-N-
glucuronide eluted at 16.5 min, and collected fractions were pooled. Evapo-
ration of the combined eluate fractions yielded 9.8 mg of white powder
(13.2%). 1H NMR (600 MHz, dimethyl sulfoxide-d6): ␦ 0.885 (t, 3H, J ϭ 7.2
Hz, CH2CH3), 2.409 (q, 2H, J ϭ 7.2 Hz, CH2CH3), 3.159–3.229 [m, 7H,
N–(CH3)2 and H-4Ј], 3.329 (m, 1H, H-3Ј), 3.464 (d, 1H, J ϭ 8.4 Hz, H-5Ј),
3.589 (m, 1H, H-2Ј), 3.843–3.886 (m, 2H, N–CH2CH2–O), 4.391 (m, 2H,
N–CH2CH2–O), 4.694 (d, 1H, J ϭ 7.2 Hz, H-1Ј),6.716 (d, 2H, J 8.4 Hz, ArH,
ortho to NCH2CH2O–), 6.80 (d, 2H, J ϭ 9 Hz, ArH, meta to NCH2CH2O–),
7.157–7.433 (m, 10H, ArH). ESI-time of flight-MS: 548.2649 [M]ϩ (error
0.18 ppm).
Incubations to Characterize Glucuronidation Kinetics in the Absence of
Modifiers. Preliminary experiments were conducted to ensure that all kinetic
determinations were performed under linear conditions with respect to time and
protein concentration. Incubation mixtures (200 ␮l final volume) contained
UGT1A4-HEK293 cell lysate (0.25 mg/ml protein for t-AND, DHT, and LTG
glucuronidation or 0.1 mg/ml protein for TAM glucuronidation), Tris-HCl buffer
(0.1 M), MgCl2 (5 mM), D-saccharic acid 1,4-lactone (5 mM), UDPGA (3 mM),
alamethicin (50 ␮g/mg protein), and DHT (3.9–250.0 ␮M), t-AND (2.8–202.2
␮M), TAM (0.5–100 ␮M), or LTG (47.4–4969.8 ␮M). DHT, t-AND, and TAM
were initially dissolved in DMSO before addition to the incubation mixtures,
whereas LTG was initially dissolved in 0.1 M acetic acid containing 4% DMSO.
The final organic solvent concentrations in all incubation mixtures were always
Յ2%. In each experiment, the organic concentration was constant irrespective of
substrate concentration. The final pH of all incubation mixtures was 7.4 at 37°C.
Cell lysates were preincubated on ice with alamethicin for 30 min before reaction
initiation. This step was followed by a 3-min preincubation at 37°C, after which
the reaction was initiated by addition of UDPGA. After a 30-min (DHT, t-AND,
and LTG) or 20-min (TAM) incubation in a shaking water bath, reactions were
terminated by addition of 200 ␮l of cold acetonitrile, followed by addition of
internal standards (DHT and t-AND glucuronidation assay: 20 ␮l of 1.07 ␮g/ml
testosterone glucuronide; TAM glucuronidation assay: 10 ␮l of 14.2 ␮g/ml lam-
otrigine glucuronide; LTG glucuronidation assay: 10 ␮l of 50 ␮g/ml morphine-
3-glucuronide). Protein precipitate was removed by centrifugation at 13,000g for
5 min, and the reaction mixture was filtered through a 0.2-␮m nylon spin filter
(Grace Davison Discovery Science, Deerfield, IL) before injection onto the HPLC
system.
existence of multiple aglycone binding sites in UGT1A4 have never
been conducted. Dihydrotestosterone (DHT) and trans-androsterone
(t-AND) (Fig. 1) are two steroidal substrates of UGT1A4. Although
the glucuronidation of DHT and t-AND by UGT1A4 has been clearly
established (Green and Tephly, 1996), a detailed kinetic analysis of
these processes has not been reported. These two compounds, based
on a planar, rigid steroidal scaffold, differ only with respect to the
position of the hydroxyl group (at position 3 or 17, the site of
glucuronidation) and the location of the ketone group (position 17 or
3). Because of the rigid steroidal scaffold shared by these two com-
pounds and the differing placement of substituents, these two com-
pounds may either occupy the same region of the active site but in
opposite orientation or occupy two separate regions in the UGT1A4
active site. Studies in our laboratory on the activities of two polymor-
phic UGT1A4 enzymes (UGT1A4.2 and UGT1A4.3) demonstrated
that mutations of amino acids in exon 1 of UGT1A4 exhibited a
differential effect on DHT and t-AND glucuronidation (J. Zhou, T. S.
Tracy, and R. P. Remmel, unpublished data). Because it is generally
accepted that aglycone substrate binding sites of UGT1A enzymes are
within the exon 1-coded N-terminal ends of the proteins (Radomin-
ska-Pandya et al., 1999), such polymorphic effects may indicate the
possibility of DHT and t-AND occupying two separate regions in
UGT1A4, reinforcing the need to conduct systematic kinetic studies
with these two compounds to explore the existence of multiple agly-
cone binding sites in UGT1A4. To this end, a detailed characterization
of the glucuronidation kinetics of these two compounds by HEK293-
expressed UGT1A4 was conducted. Interactions of DHT or t-AND
with another UGT1A4 substrate [tamoxifen (TAM) or lamotrigine
(LTG); structures shown in Fig. 1] were also evaluated.
Materials and Methods
Materials. Tamoxifen citrate and tamoxifen were purchased from MP
Biomedicals LLC (Santa Ana, CA). Lamotrigine was purchased from Toronto
Research Chemicals Inc. (North York, ON, Canada). Dihydrotestosterone,
dihydrotestosterone glucuronide, trans-androsterone (epiandrosterone), trans-
androsterone glucuronide, and testosterone glucuronide were purchased from
Steraloids (Newport, RI). Lamotrigine-N2-glucuronide was a gift from Glaxo-
SmithKline (Philadelphia, PA). UDPGA, Trizma base, Trizma HCl, D-sac-
charic acid 1,4-lactone, alamethicin, morphine-3-glucuronide, and aceto-
Incubations to Characterize Interactions between UGT1A4 Substrates.
The effect of TAM on DHT and t-AND glucuronidation was initially evaluated
FIG. 1. Structures of DHT, t-AND, TAM, and LTG. The glucuronidation sites of
the compounds are illustrated with arrows.

ATYPICAL KINETICS AND MULTIPLE BINDING SITES OF UGT1A4
433
with three DHT or t-AND concentrations (approximately 0.5 Km, Km, and 2 trans-androsterone glucuronide and testosterone glucuronide were 3.60 and
Km) and six TAM concentrations (0, 1.25, 2.5, 5, 10, and 20 ␮M). Because we 3.23, respectively. The mass spectrometer was equipped with an ESI source
observed a significant activation effect of TAM on DHT glucuronidation in
this initial study, the effect of TAM on DHT glucuronidation was further
evaluated with seven DHT concentrations (2.5–100 ␮M) in the absence or
presence of five TAM concentrations (2.5–40 ␮M). The effect of LTG on
DHT and t-AND glucuronidation was also evaluated with three DHT or t-AND
concentrations (approximately 0.5 Km, Km, and 2 Km) and six LTG concen-
trations (0, 0.375, 0.75, 1.5, 3, and 4.5 mM), and the effect of TAM on LTG
glucuronidation was studied with three LTG concentrations (0.75, 1.5, and 3
mM) and six TAM concentrations (0, 1.25, 2.5, 5, 10, and 20 ␮M). The
incubation conditions were as described above. To quantify dihydrotestoster-
one glucuronide by LC-MS, a liquid-liquid procedure was applied after reac-
tion termination and protein precipitation. Fifty microliters of 2.4 mol/l HCl
solution were added to the incubation supernatants, and the sample was
extracted twice with 500 ␮l of ethyl acetate. The ethyl acetate extracts were
then combined and dried under N2 gas. Residues were reconstituted with 50 ␮l
of water-acetonitrile (3:7, v/v) and 25 ␮l of the sample were injected onto the
HPLC system for quantification. The recovery of the liquid-liquid extraction
process was 100.2 Ϯ 6.5% for dihydrotestosterone glucuronide and 96.9 Ϯ
9.5% for the internal standard testosterone glucuronide. To study the effect of
DHT or t-AND on TAM glucuronidation, preliminary experiments were con-
ducted at three concentrations of TAM (1.51, 7.57, and 15.14 ␮M). Detailed
kinetic studies on TAM (1.0–100 ␮M) glucuronidation were conducted in the
presence of six DHT or t-AND concentrations (25–250 ␮M). The incubation
conditions were as described previously.
Chromatographic Analysis of Glucuronides. Two methods were devel-
oped to quantify trans-androsterone glucuronide and dihydrotestosterone gluc-
uronide. To characterize the glucuronidation kinetics of DHT and t-AND in the
absence of a modifier, trans-androsterone glucuronide and dihydrotestosterone
glucuronide were quantified by an LC-tandem mass spectrometry method with
an Agilent 1100 series capillary LC system coupled with a Thermo Finnigan
TSQ quantum triple quadrupole mass spectrometer (Thermo Fisher Scientific).
Separation was performed on a Thermo BetaBasic-18 column (150 ϫ 0.5 mm,
3 ␮m; Thermo Fisher Scientific). The mobile phase consisted of 10 mM
ammonium formate (A) and methanol (B) and was delivered at a flow rate of
12 ␮l/min. A linear gradient elution program, beginning with 50% of mobile
phase B and then increasing mobile phase B linearly from 50 to 90% over 1
min and holding at 90% of B for 9 min was used. The column was then
reequilibrated at initial conditions for 10 min. Both trans-androsterone gluc-
uronide and dihydrotestosterone glucuronide eluted at 7.10 min, and the
internal standard testosterone glucuronide eluted at 6.71 min. The mass spec-
trometer was equipped with an ESI interface operated in negative ion mode.
Quantification was accomplished in multiple reaction monitoring mode by
monitoring a transition pair of m/z 4653287 for trans-androsterone glucuro-
nide and dihydrotestosterone glucuronide and 4633285 for the internal stan-
dard, testosterone glucuronide. Argon was used as the collision gas. The MS
operating conditions were optimized as follows: for transandrosterone gluc-
uronide: spray voltage 4000 V, sheath gas pressure 19 mTorr, auxiliary gas
pressure 22 mTorr, capillary temperature 355°C, tube lens offset Ϫ95, colli-
sion pressure 2.2 mTorr, and collision energy 46 V; and for dihydrotestoster-
one glucuronide: spray voltage 3200 V, sheath gas pressure 19 mTorr, auxil-
iary gas pressure 5 mTorr, capillary temperature 355°C, tube lens offset Ϫ95,
collision pressure 1.9 mTorr, and collision energy 44 V. When coincubated
with a modifier, trans-androsterone glucuronide and dihydrotestosterone gluc-
uronide were quantified by a LC-MS method with an LC-MS 2010A system
(Shimadzu, Columbia, MD). Chromatographic separation was accomplished
on a Haisil C8 column (5 ␮m, 100 ϫ 2.1 mm; Higgins Analytical Inc.). For
quantitation of dihydrotestosterone glucuronide, the mobile phase consisted of
0.1% of formic acid in water (A) and acetonitrile (B) delivered at a flow rate
of 0.25 ml/min with a linear gradient elution program of 30 to 67.5% of B over
5 min, followed by an isocratic hold at 95% of B for 5 min and a 4-min column
reequilibration at the initial conditions. The retention times were 4.23 min for
operated in negative ion mode. Quantitation was accomplished in selected ion
monitoring mode by monitoring the respective [M Ϫ H]Ϫ ions: m/z ϭ 465 for
trans-androsterone glucuronide and dihydrotestosterone glucuronide and
m/z ϭ 463 for testosterone glucuronide. The MS parameters were as follows:
nebulizing gas flow 1.5 l/min; interface bias Ϫ3.50 kV; interface current
Ϫ9.20 ␮A; heating block temperature 200°C; focus lens ϩ2.5V; entrance lens
50.0 V; RF gain 5660; RF offset 5210; prerod bias ϩ4.2 V; main rod bias ϩ3.5
V; aperture Ϫ20.0 V; conversion dynode ϩ7.0 kV; detector Ϫ1.9 kV; curved
desolvation line voltage Ϫ25.0 kV; Q-array DC Ϫ35.0 V; and Q-array RF
ϩ150.0V.
Both tamoxifen-N-glucuronide and lamotrigine-N2-glucuronide were quan-
tified by LC-MS methods (LCMS-2010A; Shimadzu). Chromatographic sep-
aration was accomplished on a Haisil column (C18 5 ␮m, 100 ϫ 2.1 mm;
Higgins Analytical Inc.). The mobile phase, 0.1% formic acid (A) and 0.1%
formic in methanol (B), was delivered at a flow rate of 0.25 ml/min with the
following linear gradient elution programs: for lamotrigine-N2-glucuronide, 5
to 40% B for 5 min, 40 to 80% B for 3 min, an isocratic hold at 95% B for 3
min, and column reequilibration for 4 min (lamotrigine glucuronide eluted at
6.92 min and the internal standard morphine-3-glucuronide eluted at 2.90 min);
for tamoxifen-N-glucuronide, 5 to 40% B for 5 min, 40 to 90% B for 10 min,
an isocratic hold at 95% B for 3 min and, column re-equilibration for 4 min
(tamoxifen-N-glucuronide eluted at 15.68 min and internal standard lam-
otrigine glucuronide eluted at 5.96 min). The mass spectrometer was operated
in positive ion mode with an ESI interface. Quantification was performed in
single ion monitoring mode by monitoring m/z ϭ 432 ([M]ϩ) for lamotrigine-
N2-glucuronide, m/z ϭ 548 ([M]ϩ) for tamoxifen-N-glucuronide, and m/z ϭ
462 ([M ϩ H]ϩ) for morphine-3-glucuronide. The MS parameters were set as
follows: nebulizing gas flow 1.5 l/min; interface bias ϩ4.50 kV; interface
current 11.60 ␮A; heating block temperature 200°C; focus lens Ϫ2.5V; en-
trance lens Ϫ50.0 V; RF gain 5620; RF offset 5060; prerod bias Ϫ4.2 V; main
rod bias Ϫ3.5 V; aperture ϩ20.0 V; conversion dynode Ϫ8.0 kV; detector
Ϫ1.5 kV; curved desolvation line voltage ϩ25.0 kV; Q-array DC ϩ35.0 V;
and Q-array RF ϩ150.0 V.
Estimation of Nonspecific Protein Binding. Free fractions of DHT,
t-AND, TAM, and LTG in incubation were estimated with the Hallifax-
Houston model (eq. 1) (Hallifax and Houston, 2006), where C is protein
concentration in milligrams per milliliter and the logP values of DHT, t-AND,
TAM, and LTG are 3.428, 3.428, 6.064, and 2.04, respectively, and were
calculated with the Molinspiration-Interactive logP calculator (http://www.
molinspiration.com/services/logp.html).
1
fu ϭ
(1)
0.072⅐logP2ϩ0.067⅐logPϪ1.126
1 ϩ C ⅐ 10
Data Analysis. Glucuronidation kinetic data for each substrate in the
absence of modifiers were analyzed by fitting the Michaelis-Menten equation
(eq. 2) or an empirical uncompetitive substrate inhibition equation (eq. 3) to
the data with Sigma Plot 9.0 (Systat Software Inc., San Jose, CA) and by
nonlinear regression:
V
max ϫ ͓S͔
V0 ϭ
(2)
(3)
K
m ϩ ͓S͔
Vmax
V0 ϭ
Km ͓S͔
1 ϩ
ϩ
͓S͔ Ksi
Vmax and Km in eq. 2 were defined as the maximum velocity and substrate
concentration at which velocity is equal to half of the maximum velocity. Vmax
and Km in eq. 3 have the same definitions as in eq. 2, and Ksi is the substrate
inhibition constant. The appropriate model was selected by visual inspection of
the Eadie-Hofstee plots and comparison of the second-order Akaike informa-
dihydrotestosterone glucuronide and 3.87 min for testosterone glucuronide. tion criterion and the residual sum of squares. Kinetic parameters were esti-
For the quantitation of trans-androsterone glucuronide, the same mobile phase mated by nonlinear regression analysis with Sigma Plot 9.0.
was used, and a similar gradient elution program was applied: 30 to 60% B
over 5 min, followed by an isocratic hold at 95% B for another 5 min and a
4-min column reequilibration at the initial conditions. The retention times for
Glucuronidation kinetics in the presence of modifiers were analyzed initially by
calculating the percent rate of control (in the absence of modifiers). Modifiers that
increased or decreased glucuronidation rate by greater than 20% were considered

434
ZHOU ET AL.
to exhibit activation or inhibition effects, respectively. One-site competitive (eq. 4),
noncompetitive (eq. 5), and mixed inhibition (eq. 6) models were applied to
analyze the kinetic data, when only inhibition was observed. Vmax and Km in eqs.
4, 5, and 6 have the same definitions as above. Ki is the inhibition constant, and the
parameter ␣ reflects changes in the inhibition constant Ki. The appropriate model
was selected by visual inspection of the Dixon plots and comparison of the
second-order Akaike information criterion.
Various two-site kinetic models were applied to describe substrate inhibi-
tion kinetics as well as the interactions between TAM and DHT or TAM and
t-AND (Fig. 2; eqs. 7–11). Kinetic models with two-substrate binding sites
have been successfully used to explain substrate inhibition kinetics (Houston
and Kenworthy, 2000; Lin et al., 2001; Schrag and Wienkers, 2001). The
two-site substrate inhibition model, incorporated herein (Fig. 2A; eq. 7),
assumes one reaction site and sequential binding of substrate molecules (Ga-
letin et al., 2002). Kinetic models shown in Fig. 2, B (eq. 8) and C (eq. 9), were
used to describe the interactions between TAM and DHT. In these models,
DHT (assumed to have one binding site in UGT1A4) interacts with the
substrate inhibition site of TAM (assumed to have two binding sites in
UGT1A4). Two kinetic models (Fig. 2D; eq. 10; Fig. 2E; eq. 11) were used to
explain the effect of t-AND on TAM glucuronidation. These two models
assume that both t-AND and TAM have two binding sites in UGT1A4, and
they compete for binding to UGT1A4 at both binding sites. In Fig. 2D (eq. 10),
the reaction site of t-AND overlaps with the reaction site of TAM. In Fig. 2E
(eq. 11), the reaction site of the t-AND reaction overlaps with the substrate
inhibition site of TAM. All of the aforementioned two-site kinetic models
assume rapid equilibrium (Segel, 1993). The kinetic parameter Vmax equates to
V
max ϫ ͓S͔
V0 ϭ
(4)
(5)
(6)
͓I͔
Km
ϫ
1 ϩ
ϩ ͓S͔
ͩ ͪ
Ki
V
max ϫ ͓S͔
V0 ϭ
͓I͔
͓I͔
Km
ϫ
1 ϩ
ϩ ͓S͔ ϫ 1 ϩ
ͩ ͪ ͩ ͪ
Ki
Ki
V
max ϫ ͓S͔
V0 ϭ
͓I͔
͓I͔
Km
ϫ
1 ϩ
ϩ ͓S͔ ϫ 1 ϩ
ͩ ͪ ͩ ͪ
Ki
␣Ki
FIG. 2. Two-site kinetic models. A, a kinetic model for substrate inhibition kinetics (eq. 7). B, a kinetic model to explain the effect of TAM on DHT glucuronidation (eq.
8). C, a kinetic model to explain the effect of DHT on TAM glucuronidation (eq. 9). D and E, kinetic models to explain the effect of t-AND on TAM glucuronidation (eqs.
10 and 11). kp is the effective catalytic constant. Ks, KDHT, Kt-AND, and KTAM are binding affinity constants. Constant b and c reflect change in kp and constant d reflects
changes in binding affinity. DHTG, DHT glucuronidation; TAMG, TAM glucuronidation.

ATYPICAL KINETICS AND MULTIPLE BINDING SITES OF UGT1A4
435
kp[E]t, where [E]t is the total enzyme concentration and kp is the effective
catalytic rate constant. Ks, KDHT, Kt-AND, and KTAM are binding affinity
constants. Constants b and c reflect changes in kp. Constant d reflects changes
in binding affinity. Surface plots were generated by fitting various two-site
models to the kinetic data. Kinetic parameters were estimated with nonlinear
regression. Goodness of fit was determined by the residual sum of squares,
second-order Akaike information criterion, S.E.s of the parameter estimates
and R2.
͓DHT͔ c ⅐ ͓TAM͔ ⅐ ͓DHT͔
Vmax
⅐
ϩ
ͩ
ͪ
KDHT
d ⅐ KTAM ⅐ KDHT
V0 ϭ
(8)
2
͓DHT͔ ͓TAM͔ ͓TAM͔
͓TAM͔ ⅐ ͓DHT͔
d ⅐ KTAM ⅐ KDHT
1 ϩ
ϩ
ϩ
ϩ
2
KDHT
KTAM
KTAM
2
͓TAM͔ b ⅐ ͓TAM͔
c ⅐ ͓TAM͔ ⅐ ͓DHT͔
Vmax
⅐
ϩ
ϩ
ͩ
ͪ
2
KTAM
KTAM
d ⅐ KTAM ⅐ KDHT
V0 ϭ
(9)
2
͓DHT͔ ͓TAM͔ ͓TAM͔
͓TAM͔ ⅐ ͓DHT͔
d ⅐ KTAM ⅐ KDHT
1 ϩ
ϩ
ϩ
ϩ
2
KDHT
KTAM
KTAM
2
͓S͔ b ⅐ ͓S͔
2
Vmax
⅐
ϩ
ͩ ͪ
Ks2
͓TAM͔ b ⅐ ͓TAM͔
c ⅐ ͓TAM͔ ⅐ ͓t-AND͔
Ks
Vmax
⅐
ϩ
ϩ
ͩ
ͪ
2
V0 ϭ
(7)
2
KTAM
KTAM
d ⅐ KTAM ⅐ Kt-AND
͓S͔ ͓S͔
V0 ϭ
2
2
1 ϩ
ϩ
Ks2
͓t-AND͔ ͓t-AND͔
͓TAM͔ ͓TAM͔
2 ⅐ ͓TAM͔ ⅐ ͓DHT͔
d ⅐ KTAM ⅐ Kt-AND
Ks
1 ϩ
ϩ
ϩ
ϩ
ϩ
2
2
Kt-AND
Kt-AND
KTAM
KTAM
(10)
2
͓TAM͔ b ⅐ ͓TAM͔
c ⅐ ͓TAM͔ ⅐ ͓t-AND͔
Vmax
⅐
ϩ
ϩ
ͩ
ͪ
2
KTAM
KTAM
d ⅐ KTAM ⅐ Kt-AND
V0 ϭ
2
2
͓t-AND͔ ͓t-AND͔
͓TAM͔ ͓TAM͔
͓TAM͔ ⅐ ͓DHT͔
d ⅐ KTAM ⅐ Kt-AND
1 ϩ
ϩ
ϩ
ϩ
ϩ
2
2
Kt-AND
Kt-AND
KTAM
KTAM
(11)
Results
Nonspecific Binding of DHT, t-AND, TAM, and LTG. The
estimated free fractions of DHT and t-AND were both 81.2% in
incubations with 0.25 mg/ml protein and 91.8% at a protein concen-
tration of 0.1 mg/ml protein. The free fraction of LTG (0.25 mg/ml
protein) was estimated to be 95.2%, which is consistent with the
negligible binding of LTG to HEK293 cell lysate reported by Row-
land et al. (2006). Because the estimated nonspecific binding of DHT,
t-AND, and LTG under the incubation conditions used was less than
20%, the concentration of DHT, t-AND, and LTG added to the
incubation mixtures was not corrected for nonspecific protein binding
in calculations of kinetic parameters. However, the estimated free
fraction of TAM was 11.4% (0.1 mg/ml protein) or 4.5% (0.25 mg/ml
protein). TAM concentrations added to the incubation mixtures were
corrected for binding when kinetic parameters were estimated.
Kinetics of DHT and t-AND Glucuronidation. Initial efforts
focused on conducting a detailed evaluation of the kinetics of DHT
and t-AND glucuronidation. The Michaelis-Menten equation (eq. 2)
was fit to the data for DHT glucuronidation, whereas an empirical
uncompetitive substrate inhibition equation (eq. 3) was fit to the data
for t-AND glucuronidation. Results are presented in Fig. 3, and the
kinetic parameters obtained by nonlinear regression are presented in
Table 1. Although data for t-AND glucuronidation were not visually
different from fits with the Michaelis-Menten equation in the rate
versus [S] plot, fitting the uncompetitive substrate inhibition equation
to the data for t-AND glucuronidation generated a lower second-order
Akaike information criterion (AICc) than fitting the Michaelis-Men-
ten model to the data. [⌬AICc was 19; a value for ⌬AICc greater than
FIG. 3. Kinetic plots (rate versus [S]) for DHT (A) and t-AND (B) glucuronida-
tion by recombinant UGT1A4. The bars indicate the range of triplicate mea-
surements. The embedded figures are Eadie-Hofstee plots for the same data. The
Michaelis-Menten equation (eq. 2) was fit to the data for DHT glucuronidation.
The uncompetitive substrate inhibition equation (eq. 3) was fit to the data for
t-AND glucuronidation.
TABLE 1
Kinetic parameters for the glucuronidation of DHT, t-AND, TAM, and LTG by recombinant UGT1A4
Data are means (S.E.).
Substrate
Km
Vmax
Ksi
Kinetics Model
R2
␮M
pmol/min/mg protein
␮M
DHT
t-AND
TAM
LTG
19.6 (2.2)
23.6 (3.1)
0.90 (0.14)
1564 (126)
17.1 (0.44)
114 (7.2)
447 (37)
N.A.
514 (133)
4.6 (0.71)
N.A.
Michaelis-Menten (eq. 2)
0.9404
0.9755
0.9639
0.9926
Uncompetitive substrate inhibition (eq. 3)
Uncompetitive substrate inhibition (eq. 3)
Michaelis-Menten (eq. 2)
1064 (33)
N.A., not applicable.

436
ZHOU ET AL.
10 indicates essentially no support for the unfavorable model (Collom coincubated with a high-affinity UGT1A4 substrate, TAM. TAM, a
et al., 2008).] In addition, Eadie-Hofstee plots of each data set (Fig. 3) tertiary amine, forms a quaternary ammonium glucuronide upon
clearly demonstrated differences between the kinetic profiles of DHT UGT1A4-catalyzed N-glucuronidation. The reported Km for TAM
and t-AND glucuronidation. A two-site substrate inhibition model glucuronidation with recombinant UGT1A4 is 2.0 Ϯ 0.51 ␮M (un-
(Fig. 2A; eq. 7) was also used to describe the data for t-AND corrected for nonspecific binding) (Sun et al., 2006), which was
glucuronidation. The estimated kinetic parameters with this model are approximately 10-fold lower than the Km values for glucuronidation
presented in Table 2.
on t-AND and DHT observed in the present study, suggesting that
Effect of TAM on DHT and t-AND Glucuronidation. To test TAM may serve as a good competitive inhibitor. However, in contrast
whether differential inhibition can be observed, DHT or t-AND was with the expected competitive inhibition, TAM caused concentration-
TABLE 2
Kinetic parameters obtained by fitting various two-site models to kinetic data
Data are means (S.E.).
Substrate
Modifier
Vmax
Ksub
Kmod
b
c
d
Kinetic Model
R2
pmol/min/mg protein
␮M
␮M
t-AND
TAM
DHT
TAM
TAM
TAM
Without modifier
Without modifier
TAM
DHT
t-AND
127 (19)
625 (22)
9.8 (0.45)
562 (43)
761 (51)
761 (51)
33 (6.0)
1.4 (0.12)
18 (2.2)
1.8 (0.24)
1.3 (0.11) 106 (21)
1.3 (0.11) 106 (21)
N.A.
N.A.
0.35 (0.02)
58 (17)
0.56 (0.12)
0.12 (0.02)
N.A
0.10 (0.04)
0.18 (0.03)
0.18 (0.03)
N.A.
N.A.
8.4 (3.0)
0.52 (0.20)
0.28 (0.12)
0.14 (0.06)
N.A.
N.A.
4.4 (2.1)
2.9 (1.9)
1.2 (0.33)
0.57 (0.17)
Eq. 7
Eq. 7
Eq. 8
Eq. 9
Eq. 10
Eq. 11
0.9695
0.9712
0.9911
0.9743
0.9795
0.9795
t-AND
N.A., not applicable. Ksub, binding affinity of the substrate; Kmod, binding affinity of the modifier.
FIG. 4. Rate percentage of control versus [S] plots: for the effect of TAM on DHT glucuronidation (A); for the effect of TAM on t-AND glucuronidation (B); for the effect
of LTG on DHT glucuronidation (C); for the effect of LTG on t-AND glucuronidation (D); and for the effect of TAM on LTG glucuronidation (E). Data points are means
of duplicate measurements. Coefficients of variation are all within 10%. Symbols in A represent DHT concentrations: 2.5 (F), 5 (E), 10 (ꢀ), 20 (‚), 40 (f), 80 (Ⅺ), and
100 (ࡗ) ␮M. Symbols in B, C, and D represent DHT and t-AND concentrations: 10 (F), 20 (E), and 40 (ꢀ) ␮M. Symbols in E represent LTG concentrations: 0.75 (F),
1.5 (E), and 3.0 (ꢀ) mM. TAM concentration in the plots was corrected for nonspecific protein binding. Controls refer to incubations in which the concentration of the
modifier was zero.

ATYPICAL KINETICS AND MULTIPLE BINDING SITES OF UGT1A4
437
dependent activation/inhibition of both DHT and t-AND glucuronida- binding to the reaction site of TAM were also used to describe the
tion (Fig. 4, A and B). For DHT glucuronidation (Fig. 4A), the kinetic data, but much larger S.E.s of the parameter estimates and
maximum velocities occurred at concentrations below the highest second-order Akaike information criterion were obtained.
TAM concentration; i.e., the velocities of DHT glucuronidation ini-
Kinetics of TAM Glucuronidation. Because of the unexpected
tially increased but later decreased as the TAM concentration was effect of TAM on DHT and t-AND glucuronidation, the kinetics of
increased. In addition, the extent of the activation effect increased as TAM glucuronidation with recombinant UGT1A4 were evaluated.
the DHT concentration increased, and the greatest activation was TAM glucuronidation exhibited substrate inhibition kinetics (Fig. 6).
observed at the highest substrate concentration. Statistical comparison Both the uncompetitive substrate inhibition model (eq. 3) and a
of DHT glucuronidation in the presence and absence of 10 ␮M TAM two-site model (eq. 7) were fit to the kinetic data. The derived kinetic
(uncorrected concentration) at 40 ␮M DHT indicated that the degree parameters are presented in Tables 1 and 2, respectively. A constant
of activation by TAM was statistically significant (Student’s t test, free fraction of 11.4% for TAM was assumed in calculations of the
P Ͻ 0.001, n ϭ 6). With respect to t-AND glucuronidation (Fig. 4B), kinetic parameters.
Effect of LTG on DHT and t-AND Glucuronidation. Another
amine substrate of UGT1A4, LTG, was also evaluated as a modifier
of DHT and t-AND glucuronidation. LTG also forms a quaternary
ammonium glucuronide upon UGT1A4-catalyzed N-glucuronidation.
Initially, the kinetics of LTG glucuronidation were evaluated alone.
LTG glucuronidation exhibited a hyperbolic kinetic profile (data not
shown) with an estimated Km of 1.6 Ϯ 0.13 mM (Table 1). LTG at
concentrations ranging from ϳ0.25 Km to ϳ3 Km inhibited DHT and
t-AND glucuronidation (Fig. 4, C and D). Single-site competitive,
noncompetitive, and mixed inhibition models were evaluated to de-
scribe the inhibition data. The noncompetitive inhibition model was
associated with the lowest AICc values in both cases. The model-
predicted lines and observed data are shown in Dixon plots (Fig. 7, A
and B). The derived Ki values were 3.25 Ϯ 0.26 and 2.16 Ϯ 0.24 mM
for DHT and t-AND glucuronidation, respectively.
the activation effect of TAM was less pronounced, but features similar
to those described above were noted (Fig. 4B). The velocity of t-AND
glucuronidation initially increased but later decreased with increasing
TAM concentration and the extent of activation increased as the
t-AND concentration was increased.
To better understand the unexpected mixed effects of TAM on
DHT glucuronidation, the Michaelis-Menten model (eq. 2) was fit to
individual kinetic data sets. The kinetic parameters obtained are
shown in Table 3. Both Km and Vmax of DHT glucuronidation in-
creased as the TAM concentration was increased. In addition, simul-
taneous fitting of all kinetic data with a proposed two-site model (Fig.
2B; eq. 8) was conducted and is presented in Fig. 5. Estimated kinetic
parameters are presented in Table 2. In the two-site model (Fig. 2B;
eq. 8), DHT competes with TAM for binding to the substrate inhibi-
tion site of TAM. Models in which DHT competes with TAM for
Effects of TAM on LTG Glucuronidation. To investigate
whether TAM can activate UGT1A4 with substrates not based on the
steroidal ring structure, we also studied the effect of TAM on LTG
glucuronidation. At all TAM concentrations tested in the present
study, LTG glucuronidation was inhibited (Fig. 4E). A one-site com-
petitive inhibition model was best fit to the inhibition data (Fig. 7C).
The Ki for this interaction was 0.31 ␮M (TAM concentration was
corrected for nonspecific protein binding).
TABLE 3
Kinetic parameters for DHT glucuronidation in the presence or absence of TAM
The Michaelis-Menten equation (eq. 2) was used to fit individual kinetic data sets. Data
are means (S.E.). CLint equates to Vmax/Km; the TAM concentration was corrected for
nonspecific binding.
TAM
Km
Vmax
CLint
␮M
pmol/min/mg protein
␮l/min/mg protein
Effects of DHT and t-AND on TAM Glucuronidation. Finally,
the effects of DHT and t-AND on TAM glucuronidation were eval-
uated to assess whether the activation effects were bidirectional. Both
t-AND and DHT inhibited TAM glucuronidation in a preliminary
0
17 (1.6)
22 (2.5)
29 (3.3)
61 (4.9)
185 (32)
227 (89)
9.5 (0.3)
15 (0.60)
18 (0.79)
27 (1.1)
47 (5.7)
47 (14)
0.55
0.67
0.63
0.45
0.25
0.20
0.11
0.23
0.45
0.90
1.80
FIG. 6. Kinetic plots (rate versus [S]) for TAM glucuronidation by recombinant
UGT1A4. The bars indicate the range of triplicate measurements. The inset shows
Eadie-Hofstee plots for the same data. A two-site model (Fig. 2A; eq. 7) was fit to
the data.
FIG. 5. Kinetic modeling for the effect of TAM on DHT glucuronidation. The
surface plot was predicted with eq. 8 (Fig. 2B), and the TAM concentration in the
plot was corrected for nonspecific protein binding.

438
ZHOU ET AL.
FIG. 8. Kinetic modeling for effect of DHT (A) and t-AND (B) on TAM glucu-
ronidation. The surface plot in A is a predicted result with eq. 9 (Fig. 2C), and the
surface plot in B is a predicted result with eq. 11 (Fig. 2E). The TAM concentration
was corrected for nonspecific protein binding.
For TAM glucuronidation kinetics in the presence of t-AND, two
kinetic models (Fig. 2D; eq. 10; Fig. 2E; eq. 11) were applied to
describe the kinetic data and a similar goodness of fit was obtained.
The fit of data to eq. 11 (Fig. 2E) is illustrated in Fig. 8B.
Discussion
FIG. 7. Dixon plots for inhibition of DHT glucuronidation by LTG (A), for inhi-
bition of t-AND glucuronidation by LTG (B), and for inhibition of LTG glucu-
ronidation by TAM (C). The bars indicate the range of duplicate measurements. A
one-site noncompetitive inhibition model (eq. 5) was fit to the data in A and B.
Symbols represent DHT and t-AND concentrations: 10 (F), 20 (E), and 40 (ꢀ) ␮M.
A one-site competitive inhibition model (eq. 4) was fit to the data in C. Symbols
represent LTG concentrations: 0.75 (F), 1.5 (E), and 3.0 (ꢀ) mM. The TAM
concentration in C was corrected for nonspecific protein binding.
In the present study, DHT and t-AND (more commonly known as
epiandrosterone) were used as probe substrates to evaluate the poten-
tial existence of multiple aglycone substrate binding sites in UGT1A4.
Glucuronidation of DHT and t-AND by HEK293-expressed UGT1A4
was evaluated in the presence of another UGT1A4 substrate, TAM or
LTG. Unexpectedly, neither TAM nor LTG competitively inhibited
DHT and t-AND glucuronidation. Noncompetitive inhibition was ob-
study (data not shown). To gain further insight into the interactions of served when LTG was used as the modifier, whereas concentration-
DHT and t-AND on TAM glucuronidation, the kinetics of TAM dependent activation/ inhibition was observed with TAM as the modifier.
glucuronidation were evaluated in the presence of six concentrations These results, combined with kinetic modeling using various two-site
of DHT or t-AND. A two-site substrate inhibition model (eq. 7) was models, suggest that multiple substrate binding sites exist in UGT1A4.
applied to fit the individual kinetic data sets. Although there were no
The glucuronidation kinetics of the four UGT1A4 substrates under
clear trends of changes in the predicted kinetic parameters as DHT or investigation were carefully characterized. DHT and LTG exhibited
t-AND concentration increased, the substrate inhibition kinetic profile hyperbolic kinetics, whereas t-AND and TAM displayed substrate
of TAM glucuronidation became more hyperbolic (Fig. 8). Various inhibition kinetics. Although previous studies reported hyperbolic
two-site models were tested to simultaneously fit to the kinetic data. kinetics for TAM glucuronidation by UGT1A4 (Kaku et al., 2004;
The derived kinetic parameters are presented in Table 2. The kinetic Sun et al., 2006), there are possible explanations for this discrepancy.
model in Fig. 2C (eq. 9) adequately described the effect of DHT on In one report, a narrow TAM concentration range (1–6 ␮M, uncor-
TAM glucuronidation, and the fit of the data are presented in Fig. 8A. rected for nonspecific binding) was used, potentially precluding the

ATYPICAL KINETICS AND MULTIPLE BINDING SITES OF UGT1A4
439
observation of substrate inhibition at higher TAM concentrations (Sun Assuming the same binding scenario as in Fig. 2B (eq. 8), the
et al., 2006). In the second case, a 1-h incubation was conducted kinetic model in Fig. 2C (eq. 9) adequately explained the effect of
(Kaku et al., 2004), suggesting that linear incubation conditions may DHT on TAM glucuronidation. But in this case, the predicted c value
not have been operational. Our preliminary studies to determine is less than 1, indicating that the DHT-E-TAM complex is less
linearity with incubation time and protein concentration for TAM productive than the E-TAM complex, consistent with the observed
glucuronidation indicated that a low protein concentration and short inhibition effect of DHT on TAM glucuronidation. In addition, in this
incubation time were required to maintain steady-state conditions.
model TAM substrate inhibition kinetics would be eliminated as
In the present study, TAM and t-AND substrate inhibition were DHT-E-TAM becomes the dominant productive complex, also con-
described with a two-site model, as depicted in Fig. 2A (eq. 7). In both sistent with our observation that the substrate inhibition kinetic profile
cases, the estimated b values were less than 1, indicating that the SES of TAM glucuronidation became more hyperbolic as the DHT con-
complex is less productive than the ES complex. In addition, consis- centration was increased.
tent with the more pronounced substrate inhibition of TAM glucu-
ronidation, the estimated b value for TAM glucuronidation is smaller t-AND glucuronidation as on DHT glucuronidation: concentration-
than the b value obtained for t-AND glucuronidation. dependent activation/inhibition and greater activation at higher sub-
Although the activation is modest, TAM exhibits the same effect on
For UGT-catalyzed glucuronidation, substrate inhibition kinetics strate concentrations. However, the kinetic model in Fig. 2B (eq. 9)
can also be explained by the aglycone substrate binding to the en- may not adequately explain the interactions of TAM on t-AND
zyme-UDP complex, resulting in a nonproductive dead-end complex glucuronidation because of the substrate inhibition kinetics of t-AND
(Luukkanen et al., 2005). However, such a mechanism in which only (two t-AND binding sites). Kinetic models in which t-AND and TAM
one aglycone substrate binding site is incorporated does not ade- both have two binding sites on UGT1A4 may be applicable. Kinetic
quately explain the activation effect of TAM on DHT and t-AND modeling with more data points than obtained in Fig. 4B is needed to
glucuronidation. UDP, a product of catalysis, has been reported to be adequately characterize the effect of TAM on t-AND glucuronidation.
an inhibitor for UGT1A4 (IC50 ϭ 31 ␮M) (Fujiwara et al., 2008). It
Kinetic studies to characterize the effect of t-AND on TAM glu-
is also possible that the observed substrate inhibition is due to the curonidation were carefully conducted. Two kinetic models (Fig. 2D;
increased amount of UDP formation at high substrate concentrations. eq. 10; Fig. 2E; eq. 11) in which the two binding sites of t-AND
However, the calculated maximum UDP concentration was ϳ0.5 ␮M overlap with the two binding sites of TAM adequately explained the
in our study. Thus, the inhibition of UGT1A4 by UDP should be kinetic data. Again, the predicted c values (less than 1) are consistent
negligible under the incubation conditions used herein.
with the inhibition effect of t-AND on TAM glucuronidation and
A few cases of heteroactivation have been reported with UGTs TAM substrate inhibition kinetics being eliminated as the less pro-
(Williams et al., 2002; Mano et al., 2004; Pfeiffer et al., 2005; ductive t-AND-E-TAM complex becomes the dominant productive
Uchaipichat et al., 2008; Hyland et al., 2009). In the present study, complex in the models is consistent with our observation.
TAM both activated and inhibited DHT glucuronidation in a concen-
The unexpected heteroactivation on DHT and t-AND glucuronida-
tration-dependent fashion. A two-site kinetic model (Fig. 2B; eq. 8), tion by TAM led us to investigate the effect of TAM on UGT1A4
which considers the kinetic properties of DHT and TAM, adequately activity with a different type of UGT1A4 substrate: LTG (an aromatic
explained the effect of TAM on DHT glucuronidation. In this model, amine substrate of UGT1A4). In contrast to the concentration-depen-
the overall effect of TAM is controlled by three enzyme-associated dent activation/inhibition on DHT and t-AND glucuronidation, LTG
complexes (E-TAM, TAM-E-TAM, and TAM-E-DHT). Complexes N-glucuronidation was competitively inhibited by TAM, suggesting
E-TAM and TAM-E-TAM are not productive. The presence of these that the heteroactivation of UGT1A4-catalyzed glucuronidation by
complexes results in less enzyme available for association with the TAM is substrate-dependent.
substrate (DHT), producing an inhibition effect. However, the DHT-
LTG was also evaluated as a modifier of DHT and t-AND glucu-
E-TAM complex is more productive than the DHT-E complex (c ϭ ronidation. LTG inhibited both t-AND and DHT glucuronidation, but
8.36). The presence of the DHT-E-TAM complex leads to activation. interestingly the one-site noncompetitive inhibition model (eq. 5)
At low TAM concentrations, the activation resulting from the pres- better described the kinetic data than the one-site competitive inhibi-
ence of the DHT-E-TAM complex overcomes the inhibition effect, tion model (eq. 4). The observed noncompetitive inhibition of DHT
resulting in an overall activation effect. At high TAM concentrations, glucuronidation by LTG suggests that these two UGT1A4 substrates
the TAM-E-TAM complex becomes the dominant form for TAM have distinct binding sites within the active site of UGT1A4, assum-
associating with the enzyme, resulting in an overall inhibition effect. ing each has only one binding site.
Also interestingly, in contrast with most previous reports of enzyme
The present study provides compelling evidence for the existence
heteroactivation, in which the extent of heteroactivation decreases as of at least two aglycone binding sites in UGT1A4. Although models
the substrate concentration increases (Hutzler et al., 2001; Kenworthy can be developed to describe the kinetic data, additional biophysical/
et al., 2001; Uchaipichat et al., 2008), DHT glucuronidation is in- biochemical studies are needed to delineate the specific binding
creasingly heteroactivated by TAM as the substrate (DHT) concen- region(s) of each molecule in UGT1A4. Additional kinetic studies
tration was increased. This discrepancy is probably due to different with a wider range of UGT1A4 substrates are also needed to evaluate
mechanisms of heteroactivation. In previous cases, heteroactivation the range of substrates for which atypical kinetics are operable.
was largely due to the positive cooperative binding of substrates and UGT1A4 has been reported to form homodimers (Operana and Tukey,
modifiers to the enzyme (Hutzler et al., 2001; Kenworthy et al., 2001; 2007), which may also play a role in these atypical kinetic phenom-
Uchaipichat et al., 2008). However, in the present study, the increased ena. It is yet to be determined whether the two aglycone binding sites
glucuronidation appeared to be due to the presence of a more produc- exist in different monomers or whether each monomer has two sep-
tive modifier-E-substrate complex (DHT-E-TAM) (c ϭ 8.36). The arate aglycone binding sites.
percentage of the DHT-E-TAM complex among all enzyme com-
In vitro-in vivo extrapolations for UGT-catalyzed metabolism have
plexes is greater at high substrate concentrations than at low substrate proven problematic for a number of reasons, including an inability to
concentrations and therefore more activation was observed at high estimate in vivo UGT enzyme amounts, lack of isoform-specific probe
substrate concentrations.
substrates and inhibitors, overlapping substrate specificities (Miners et

440
ZHOU ET AL.
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Miners JO, Knights KM, Houston JB, and Mackenzie PI (2006) In vitro-in vivo correlation for
drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises.
Biochem Pharmacol 71:1531–1539.
Miners JO, Smith PA, Sorich MJ, McKinnon RA, and Mackenzie PI (2004) Predicting human
drug glucuronidation parameters: application of in vitro and in silico modeling approaches.
Annu Rev Pharmacol Toxicol 44:1–25.
Ohno S, Kawana K, and Nakajin S (2008) Contribution of UDP-glucuronosyltransferase 1A1 and
1A8 to morphine-6-glucuronidation and its kinetic properties. Drug Metab Dispos 36:688–
694.
Operan˜a TN and Tukey RH (2007) Oligomerization of the UDP-glucuronosyltransferase 1A
proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer
and co-immunoprecipitation. J Biol Chem 282:4821–4829.
Pfeiffer E, Treiling CR, Hoehle SI, and Metzler M (2005) Isoflavones modulate the glucuronida-
tion of estradiol in human liver microsomes. Carcinogenesis 26:2172–2178.
Radominska-Pandya A, Czernik PJ, Little JM, Battaglia E, and Mackenzie PI (1999) Structural
and functional studies of UDP-glucuronosyltransferases. Drug Metab Rev 31:817–899.
Rios GR and Tephly TR (2002) Inhibition and active sites of UDP-glucuronosyltransferases 2B7
and 1A1. Drug Metab Dispos 30:1364–1367.
Rowland A, Elliot DJ, Knights KM, Mackenzie PI, and Miners JO (2008) The “albumin effect”
and in vitro-in vivo extrapolation: sequestration of long-chain unsaturated fatty acids enhances
phenytoin hydroxylation by human liver microsomal and recombinant cytochrome P450 2C9.
Drug Metab Dispos 36:870–877.
Rowland A, Elliot DJ, Williams JA, Mackenzie PI, Dickinson RG, and Miners JO (2006) In vitro
characterization of lamotrigine N2-glucuronidation and the lamotrigine-valproic acid interac-
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al., 2004, 2006), and the “albumin effect” (Rowland et al., 2008).
Accumulating evidence from the current study and others referenced
above suggests that atypical kinetics involving this enzyme family
may also contribute to the difficulty in making in vitro-in vivo
correlations. Atypical kinetic profiles, such as the substrate inhibition
observed in the present study, complicate the estimation of intrinsic
clearance. In addition, the presence of multiple aglycone binding sites
and the substrate-dependent heteroactivation as observed in the
present study, complicate the prediction of drug interactions. In sum-
mary, the present study reinforces the need for careful characterization
of UGT1A4 kinetics and highlights the caveats of making in vitro-in
vivo correlations with this important metabolizing enzyme. For the
purpose of screening for UGT1A4 inhibitors, the present study sug-
gests the potential need to use multiple probe substrates.
Acknowledgments. We thank Dr. Philip Lazarus at Penn State
University for providing transfected HEK-293 cells that express
UGT1A4. Synthesis of tamoxifen-N-glucuronide was done with the
help of Dr. Courtney Aldrich, Center for Drug Design, University of
Minnesota. Morphine-3-glucuronide was a gift from Dr. Cheryl Zim-
merman, University of Minnesota.
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