UDP-glucuronic acid binds first and the aglycone substrate binds second to form a ternary complex in UGT1A9-catalyzed reactions, in both the presence and absence of bovine serum albumin

Article abstract of DOI:10.1124/dmd.112.047746

The presence of bovine serum albumin (BSA) largely modulates the enzyme kinetics parameters of the human UDP-glucuronosyltransferase (UGT) 1A9, increasing both the apparent aglycone substrate affinity of the enzyme and its limiting reaction velocity (Drug Metab Dispos 39:2117-2129, 2011). For a better understanding of the BSA effects and an examination of whether its presence changes the catalytic mechanism, we have studied the enzyme kinetics of 4-methylumbelliferone glucuronidation by UGT1A9 in the presence and absence of 0.1% BSA, using bisubstrate enzyme kinetic experiments, in both the forward and reverse directions, as well as product and dead-end inhibition. The combined results strongly suggest that the reaction mechanism of UGT1A9, and presumably other human UGTs as well, involves the formation of a compulsory-order ternary-complex, with UDP-α-D-glucuronic acid (UDPGA) as the first binding substrate. Based on the enzyme kinetic parameters measured for the forward and reverse reactions, the equilibrium constant of the overall reaction was calculated (Keq = 574) and the relative magnitudes of the reaction rate constants were elucidated. The inclusion of BSA in the bisubstrate kinetic experiments quantitatively changed the apparent enzyme kinetic parameters, presumably by removing internal inhibitors that bind to the binary enzyme-UDPGA (E-UDPGA) complex, as well as to the ternary E-UDPGA-aglycone complex. Nevertheless, the underlying compulsory-order ternary-complex mechanism with UDPGA binding first is the same in both the absence and presence of BSA. The results offer a novel understanding of UGT enzyme kinetic mechanism and BSA effects. Copyright

Full text of DOI:10.1124/dmd.112.047746

DMD Fast Forward. Published on August 21, 2012 as doi:10.1124/dmd.112.047746
Title page
UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a
Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of
BSA
Nenad Manevski, Jari Yli-Kauhaluoma and Moshe Finel
Division of Pharmaceutical Chemistry (N.M. and J.Y.-K.), and Centre for Drug Research
(
N.M., and M.F.), Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.

Running title page
Running title: Enzyme kinetics of UGT1A9 in the presence of BSA
Corresponding author: Dr. Moshe Finel, Centre for Drug Research, Faculty of Pharmacy,
P.O. Box 56 (Viikinkaari 5), FI-00014 University of Helsinki, Finland
E-mail: Moshe.Finel@helsinki.fi, Fax: +358 9 191 59556
Manuscript statistics:
Number of text pages: 40
Number of tables: 5
Number of figures: 8
Number of references: 41
Number of words in the Abstract: 241
Number of words in the Introduction: 742
Number of words in the Discussion: 1577
Abbreviations:
BSA, bovine serum albumin; CI, confidence interval of the mean; Eq., equation; 4-MU, 4-
methylumbelliferone; 4-MUG, 4-methylumbelliferone- -D-glucuronide; RED, rapid
equilibrium dialysis; UDPGA, UDP- -D-glucuronic acid; UGT, UDP-glucuronosyltransferase.

Abstract
The presence of bovine serum albumin (BSA) largely modulates the enzyme kinetics
parameters of the human UDP-glucuronosyltransferase (UGT) 1A9, increasing both the
apparent aglycone substrate affinity of the enzyme and its limiting reaction velocity (Manevski
et al., Drug Metabol. Dispos. 39:2117–2129, 2011). For better understanding of the BSA
effects and examining whether or not its presence changes the catalytic mechanism, we have
studied the enzyme kinetics of 4-methylumbelliferone (4-MU) glucuronidation by UGT1A9 in
the presence and absence of 0.1% BSA, using bisubstrate enzyme kinetic experiments, in both
the forward and reverse directions, as well as product and dead-end inhibition. The combined
results strongly suggest that the reaction mechanism of UGT1A9 and, presumably, other
human UGTs as well, involves the formation of a compulsory-order ternary-complex, with
UDPGA as the first binding substrate. Based on the enzyme kinetic parameters measured for
the forward and reverse reactions, the equilibrium constant of the overall reaction was
calculated (Keq = 574) and the relative magnitudes of the reaction rate constants were
elucidated. The inclusion of BSA in the bisubstrate kinetic experiments quantitatively changed
the apparent enzyme kinetic parameters, presumably by removing internal inhibitors that bind
to the binary E•UDPGA complex, as well as to the ternary E•UDPGA•aglycone complex.
Nevertheless, the underlying compulsory-order ternary-complex mechanism with UDPGA
binding first is the same in both the absence and presence of BSA. The results offer novel
understanding of UGT enzyme kinetic mechanism and BSA effects.

Introduction
Human UDP-glucuronosyltransferases (UGTs) transfer the glucuronic acid moiety from UDP-
-D-glucuronic acid (UDPGA) to nucleophilic groups on small organic molecules of both
endo- and xenobiotic origin (Bock, 2010, Miners et al., 2010). The conjugation with
glucuronic acid increases the hydrophilicity of the original aglycone substrate and stimulates
excretion from the cell, probably by facilitating molecular recognition by efflux transporters
(
Jemnitz et al., 2010). The amino acid sequences of individual UGT enzymes are highly
homologous (Mackenzie et al., 2005), and the enzymes exhibit distinct but partially
overlapping substrates and inhibitors specificity (Miners et al., 2010). The UGTs are variably
expressed in human tissues, particularly in the liver, intestine, and kidneys (Court et al., 2012,
Ohno and Nakajin, 2009). The enzyme at the focus of this study, UGT1A9, is important in the
metabolic elimination of several therapeutic drugs, including entacapone (Lautala et al., 2000),
propofol (Soars et al., 2004), and mycophenolic acid (Bernard and Guillemette, 2004), and is
highly expressed in liver and kidneys (Court et al., 2012).
The presence of purified bovine serum albumin (BSA) in the glucuronidation reaction greatly
enhances the in vitro activity of the human UGT1A9; regardless of whether recombinant
enzyme or human liver microsomes (HLM) are used as the enzyme source (Manevski et al.,
2
011). The inclusion of BSA in both the entacapone and 4-methylumbelliferone (4-MU)
glucuronidation assays decreased the K
m
values, increased the Vmax values, and led to some
changes in the enzyme kinetic model (Manevski et al., 2011). Such changes in enzyme
kinetics open many questions, both practical and theoretical. Two of the more challenging
questions are what is the underlying mechanism of the BSA effects and are the results of
earlier studies on the UGTs’ reaction mechanism—performed in the absence of BSA—also
valid in the presence of this modulator.

The previously suggested BSA-mediated removal of competitive inhibitors, perhaps fatty
acids that are released upon cell disruption, could account for the observed K decrease that
m
was found in UGT2B7 (Rowland et al., 2007, Rowland et al., 2008). Nevertheless, the
removal of competitive inhibitors could not explain the Vmax increase or the changes in the
enzyme kinetic model that we found in UGT1A9 (Manevski et al., 2011). To account for the
latter observations, we proposed that BSA also removes inhibitors that are not directly
competing with aglycone substrate binding (Manevski et al., 2011). Such a hypothesis,
however, is poorly defined without knowledge of the reaction mechanism, particularly in
enzymes, like the UGTs, that catalyze a two-substrate two-product reaction.
The UGTs reaction mechanism has been studied extensively in the last 40 years, yielding
variable results (Table 1). Many of these studies found that, regardless of enzyme source and
experimental conditions, UGT-catalyzed reactions follow ternary-complex mechanism. The
formation of ternary-complex is also supported by the evidence that the glucuronidation
reaction resembles S
2-type nucleophilic substitution reaction, with the inversion of the
N
glucuronic acid anomeric carbon from - in UDPGA to -configuration in the formed
glucuronide (Axelrod et al., 1958, Johnson and Fenselau, 1978). Alongside the broad
agreement on the formation of ternary complex, however, there is disagreement among the
previous studies on whether or not the two substrates, the aglycone substrate and the UDPGA,
bind in a random order or in a compulsory order (Table 1). The observed discrepancies in the
order of substrate binding may have arisen from the use of liver microsomal preparations that
contain multiple UGT enzymes (Potrepka and Spratt, 1972, Vessey and Zakim, 1972, Sanchez
and Tephly, 1975, Rao et al., 1976, Koster and Noordhoek, 1983), or of partially purified UGT
enzymes (Matern et al., 1982, Matern et al., 1982, Falany et al., 1987, Yin et al., 1994) that
may have been inactivated by detergents during the purification process (Kurkela et al., 2003).

A previous study from this laboratory, that was performed using individual recombinant UGT
enzymes of subfamily 1A, concluded that UDPGA is the first binding substrate (Luukkanen et
al., 2005). That study was carried out in the absence of BSA and focused on UGT1A9, an
enzyme we recently found to be highly affected by the presence of BSA (Manevski et al.,
2
011). Due to this, it is particularly interesting to examine whether or not the presence of BSA
affects the reaction mechanism of UGT1A9. In order to address this question, we have carried
out the detailed study that is described below. The results provide comprehensive insights into
the UGT reaction mechanism, as well as a theoretical framework for future mechanistic
studies with recombinant UGTs.

Materials and Methods
Compounds and reagents. 4-MU (>99.0%, CAS 90-33-5), 1-naphthol (>99%, CAS 90-15-3),
UDPGA (ammonium salt, 98–100%, CAS 43195-60-4), UDP (disodium salt hydrate, 96%,
CAS 27821-45-0), 4-methylumbelliferone- -D-glucuronide (4-MUG, 98%, CAS 6160-80-1),
1
-naphthol- -D-glucuronide (>99%, CAS 83833-12-9), sodium phosphate monobasic
dihydrate ( 99%, CAS 13472-35-0), and bovine serum albumin (BSA, 96%, CAS 9048-46-
, essentially fatty acid free, 0.004%) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Magnesium chloride hexahydrate and perchloric acid were obtained from Merck
Darmstadt, Germany). Formic acid (98–100%) was from Riedel-deHaën (Seelze, Germany).
8
(
Disodium hydrogen phosphate dihydrate was purchased from Fluka (Buchs, Switzerland).
Liquid chromatography–mass spectrometry-grade solvents were used throughout the study.
Recombinant UGT1A9 and negative control membranes. Recombinant human UGT1A9
was expressed, as His-tagged protein, in baculovirus-infected Sf9 insect cells (Kurkela et al.,
2
007). Negative control membranes, called "Bac control", were prepared from insect cells that
were infected with a modified baculovirus that does not encode any functional UGT. Protein
concentration in the UGT1A9-enriched and "Bac control" membranes was determined by the
BCA method (Pierce Biotechnology, Rockford, IL, USA).
Substrate binding assays. The binding of entacapone, 4-MU, and 1-naphthol to 0.1% and 1%
BSA, as well as to insect cells membranes, the "Bac control" membranes, was measured in this
study by the rapid equilibrium dialysis system (RED, Thermo Scientific, Rockford, IL, USA).
®
The system consisted of reusable Teflon base plate and single-use device insets comprised of
two side-by-side vertical cylinder chambers separated by dialysis membrane (molecular
weight cut-off value ∼ 8000 Da). The RED device insets were placed into the base plate and
the sample chamber was filled with 400 µL of the test compound solution in 50 mM phosphate

buffer (pH 7.4) that also contained the “binding” macromolecules, 0.1%, 1% BSA or insect
cell membranes (0.02–0.2 mg/mL of total membrane protein). The buffer chamber was filled
only with 600 µL of blank buffer, 50 mM phosphate buffer (pH 7.4), without the test
compound and the binding macromolecule. The filled up RED device was covered with
®
Parafilm and incubated at 100 rpm at 37 °C for 6 to 8 h. The test compounds concentration
range in the sample chamber was 5–750 µM, 5–500 µM and 0.05–5 µM for entacapone, 4-
MU, and 1-naphthol, respectively. Following incubation, a 50 µL aliquot from each chamber
was mixed with 100 µL of ice-cold 4 M perchloric acid/methanol (1:5 mix), placed at –18 °C
for 30 min, centrifuged (at room temperature) for 5 min at 16000g and the test compound
concentration in the resulting supernatant was determined by HPLC analyses. The unbound
fraction of the test compound, f
u
, was calculated using the equation:
,
where [S]buffer chamber and [S]sample chamber are the concentrations of the test compound in the
respective chambers at the end of the incubation.
Glucuronidation assays. Stock solutions of 4-MU, 4-methylumbelliferone- -D-glucuronide
(4-MUG), and 1-naphthol were prepared in methanol and diluted with methanol to the desired
concentrations immediately before use. Appropriate amounts of these dilutions were
transferred into 1.5 mL bench-top centrifuge tubes and the solvent was evaporated in vacuo at
ambient temperature. The solid residues were dissolved in the reaction mixture that was
composed of phosphate buffer (50 mM, pH 7.4), MgCl
the enzyme source (0.02–0.2 mg/mL of total membrane protein, either UGT1A9-enriched or
Bac control" membranes), to a final volume of 100 µL. The concentration of MgCl required
2
(10 mM), BSA (zero or 0.1%), and
"
2
for optimal UGT1A9 activity was selected based on literature data (Walsky et al., 2012).
Alamethicin was not added since it does not significantly stimulate the glucuronidation

activity of recombinant UGT enzymes expressed in Sf9 insect cells (Walsky et al., 2012,
Zhang et al., 2011, Kaivosaari et al., 2008).
The reaction mixtures, with the dissolved aglycone substrate, were incubated first for 20 min
at room temperature and then at 37°C for 5 min. The glucuronidation reaction was initiated by
the addition of UDPGA (to a final concentration of 20–5000 µM), and carried out, protected
from light, at 37°C for 15–60 min, depending on the substrate consumption rate. The reactions
in the reverse direction were initiated by the addition of UDP to a final concentration of 5–
1
000 µM. Negative controls, including incubations in the absence of UDPGA (or UDP),
without substrate, or with the “Bac-control” insect cells membranes instead of the UGT1A9-
enriched membranes and in the presence of both substrates, were carried out for each set of
assays. The glucuronidation reactions in the forward direction were terminated by the addition
of 60 µL ice-cold 4 M perchloric acid/methanol (1:5). To minimize the risk of non-enzymatic
glucuronide hydrolysis, the reactions in the reverse direction were terminated by the addition
of 100 µL ice-cold 5% acetic acid in methanol. Following reaction termination, the tubes were
transferred to –20 °C for 60 min and then centrifuged at 16000g for 5 min at room
temperature. Aliquots of the resulting supernatants were transferred to dark glass vials and
subjected to HPLC analyses.
Analytical methods. The HPLC system consisted of an Agilent 1100 series degasser, binary
pump, 100-vial autosampler, thermostated column compartment, multiple wavelengths UV
detector, and fluorescence detector (Agilent Technologies, Palo Alto, CA, USA). A Poroshell
1
20 EC-C18 column (4.6 × 100 mm, 2.7 µm; Agilent Technologies, Palo Alto, CA, USA),
was used for all the separations, at column temperature of 40°C and an eluent flow rate of 1
mL/min. The resulting chromatograms were analyzed with Agilent ChemStation software (rev.

B.01.01) on Windows XP Professional. Further details of the analytical methods and analyte
quantification are presented in Table 2.
Enzyme kinetic assays and data analysis. The protein concentrations and incubation times
for the kinetic analyses reactions were selected based on preliminary assays so that no more
than 10% of the substrate is consumed during the incubation. In the case of bisubstrate enzyme
kinetics and inhibition assays, we performed a number of optimization assays in order to select
the suitable concentrations of substrates and inhibitors, as well as protein concentration,
incubation time and optimal parameters of the analytical method for the final assays. Initial
velocity measurements in the enzyme kinetic assays in which a concentration of a single
substrate was varied were performed in triplicates and are presented as the average ± S.E.
Initial velocity measurements for the bisubstrate enzyme kinetics and the inhibition studies
were performed in duplicates and are presented as the average value (the variation between
two parallel samples in these experiments was not more than 15%, but the differences between
the duplicates were much lower than this in most cases). The lines in the Eadie-Hofstee and
Lineweaver-Burk plots were plotted with enzyme kinetic parameters obtained from the curve
fittings, not by linear regression. Although Eadie-Hofstee plots offer a more reliable
presentation of the enzyme kinetics data (Dowd and Riggs, 1965), due to the widespread use
of the Lineweaver-Burk “double-reciprocal” plots, we have used them to visualize the
outcome of product and dead-end inhibition studies. The bisubstrate experiments initial rates
for the forward reaction catalyzed by the recombinant UGT1A9 were determined by varying
the concentration of both substrates simultaneously: the sugar acid donor UDPGA (20–2000
µM) and the aglycone substrate (1–50 and 1–180 µM, with and without substrate inhibition,
respectively). The initial rates in the bisubstrate reverse reactions were measured by
simultaneously varying the concentration of UDP (5–1000 µM) and 4-MUG (150–10000 µM).
The incubation times were varied from 10 to 60 min. The enzyme kinetic parameters were

obtained by fitting the kinetic models to the experimental data, using GraphPad Prism version
.04 for Windows (GraphPad Software Inc., San Diego, CA, USA). The best (most
5
appropriate for each reaction) kinetic model was selected based on visual inspection of the
Eadie-Hofstee and Lineweaver-Burk plots, residuals graphs, parameter S.E. and 95%
2
confidence intervals estimates (95% CI), the calculated r values, and the corrected Akaike’s
information criterion. In assays containing BSA, the free substrate or inhibitor concentrations
u
(f , or fraction unbound), were corrected according to the measured drug binding to BSA under
the specific conditions of each glucuronidation assay (see Drug binding assays above). The
initial glucuronidation velocities in the single substrate enzyme kinetics assays were fitted to
the following equations:
Michaelis-Menten model:
(
1)
Where v is the initial velocity of the reaction, [S] is the substrate concentration, Vmax is the
limiting velocity at saturating concentration of substrate, and K
m
is the Michaelis-Menten
constant (concentration of substrate at 0.5 of Vmax).
Substrate inhibition model:
(
2)
Where Ksi is the constant describing the substrate inhibition interaction. This model is based
on mechanism in which a second molecule of substrate binds to preformed enzyme-substrate
complex (E•S) and thus acts as uncompetitive inhibitor of the reaction.

For the calculation of enzyme inhibition constants, the obtained data were fitted to the models
for competitive (eq. 3), mixed-type (eq. 4), noncompetitive (eq. 5), and uncompetitive
inhibition (eq. 6) (Copeland, 2005):
(3)
(4)
(5)
(6)
Where Kic, Kim, Kin, and Kiu are the competitive, mixed-type, noncompetitive, and
uncompetitive inhibition constants, respectively. The coefficient in eqs. 4 and 6 represents
the relative difference in inhibitor’s binding affinity between the free enzyme (competitive
modality) and the E•S complex (uncompetitive modality).
The reversible transfer of glucuronic acid from UDPGA to the aglycone substrate is described
in this study by the general reaction: AX + B
BX + A, in which AX, B, BX, and A
represent UDPGA, the aglycone substrate, the glucuronide conjugate and UDP, respectively.
For the analysis of bisubstrate enzyme kinetic data, the initial velocities were fitted to the
following equations:
Compulsory-order ternary-complex model based on a steady-state assumption (Cornish-
Bowden, 2012):
(
7)

Where [AX] and [B] are the concentrations of UDPGA and aglycone substrate, respectively,
max is t he limiting velocity at saturating concentration of both AX and B, KiAX is the
equilibrium dissociation constant for the E + A X E•AX reaction, KmAX is the limiting
V
Michaelis constant for AX when B is saturating, and KmB is the limiting Michaelis constant for
B when AX is saturating. This model assumes that substrate AX binds first and B is the
second-binding substrate. In the case of the reverse reaction, UDP (A) and 4-MUG (BX) are
considered as the first and second substrates, respectively (Fig. 8). The Haldane relationship
for the compulsory-order ternary-complex mechanism is gi ven by
,
where
and
are the limiting velocities in the forward and reverse
direction, respectively.
Compulsory-order ternary-complex model based on a steady-state assumption with
substrate inhibition (Cornish-Bowden, 2012):
(
8)
Where KsiB is a constant that describes the substrate inhibition interaction. This model assumes
that AX and B ar e first and second enzyme substrates, respectively, and that substrate
inhibition arises from binding of the second substrate, B, to the “wrong” binary complex, E•A,
instead of E•AX (see Fig. 8).
Compulsory-order ternary-complex model based on a rapid-equilibrium assumption
(
Alberty, 2011, Copeland , 2000):
(
9)

Where KsAX is the equilibrium dissociation constant for the E•AX complex (E + AX E•AX)
and KsB is th e equilibrium dissociation constant for the E•AX•B complex (E•AX + B
E•AX•B). This model assumes that AX and B are the first and second binding substrates,
respectively.
Random-order ternary-complex model based on a rapid-equilibrium assumption
(
Alberty, 2011):
(
10)
Where KiAX and KiB are the equilibrium dissociation constants for the E•AX and E•B
complexes, respectively. K is t he first equilibrium dissociation constant for the E•AX•B
complex (E•AX•B E•AX + B). KA, the second equilibrium dissociation constant for the
E•AX•B complex (E•AX•B E•B + AX), is not included in this equation but can be
calculated from the following relationship: KiA
B
B A
K = KiBK .
Substituted-enzyme (ping-pong bi-bi) model based on a steady-state assumption
(
Cornish-Bowden, 2012):
(
11)
Where KmAX and KmB are the limiting Michaelis constants for AX and B, as in eq. 7. This
model also implicitly assumes mechanistically reasonable compulsory-order mechanism,
where AX and B are the first and second binding substrates, respectively.
Substituted-enzyme (ping-pong bi-bi) model based on a steady-state assumption with
substrate inhibition (Cornish-Bowden, 2012):
(
12)

In this model substrate inhibition arises from B binding to the free enzyme (E + B E•B) and
siB represents the dissociation constant for the E•B complex.
K

Results
Substrates binding to BSA and insect cell membranes. The binding of entacapone, 4-MU,
and 1-naphthol to BSA, both 0.1% and 1%, and to insect cell membranes, the "Bac control"
membranes that lack any human UGT, was measured in this study by the RED system. The
results of the binding assays with this system, in the way we performed them (see Materials
and Methods for technical details) were in good agreement with the binding results for
entacapone and 4-MU that we previously obtained using ultrafiltration (Manevski et al., 2011)
(Fig. 1). For all the tested compounds in this study, the equilibrium was reached within 8 h of
incubation in the RED device, under constant shaking. Overnight incubation seemed more
susceptible to inaccuracies due to volume shifts, caused by hydrostatic pressure, and increased
risk of compounds instability (results not shown). The nonspecific binding to the RED device
of the tested compounds was low ( 20%).
Entacapone binds strongly to 0.1% BSA and, as previously reported (Manevski et al., 2011),
the measured f for this drug exhibits hyperbolic saturable binding profile (Fig. 1A). The 4-
u
MU binding to 0.1 and 1% BSA was partially concentration dependent (Fig. 1B), as
previously found using the ultrafiltration method (Manevski et al., 2011). The binding of 1-
naphthol to 0.1% BSA was rather low and concentration independent,
average ± S.E.; Fig. 1C).
0.78 ± 0.01
(
The results of the binding experiments of entacapone, 4-MU, and 1-naphthol to insect cells
membranes revealed that in the presence of up to 0.2 mg/mL (total protein), the highest protein
concentration used in this work, the binding of all the tested compounds was low. The binding
of UDPGA, UDP, and 4-MUG to 0.1% BSA was negligible (data not shown).
BSA effects on the enzyme kinetics of 1-naphthol glucuronidation by UGT1A9. We have
examined 1-naphthol glucuronidation by UGT1A9 since, for the purpose of this study, it is a

high affinity but low turnover substrate that can serve as an inhibitor for the aglycone substrate
binding (Luukkanen et al., 2005). The question we tried to answer in this experiment was
whether or not the (low rate) glucuronidation of 1-naphthol by UGT1A9, as well as the
usefulness of this compound as inhibitor for UGT1A9, are affected by the presence of BSA in
the reaction medium. The addition of 0.1% BSA affected the enzyme kinetics of 1-naphthol
glucuronidation by UGT1A9 in a similar manner to its effect on th e glucuronidation of
entacapone and 4-MU by UGT1A9, namely K
011). It lowered the K of UGT1A9 in the 1-naphthol glucuronidation reaction from 376 ± 36
CI 95%, 302–449) to 177 ± 14 nM (CI 95%, 149–206) and increased the reaction’s Vmax from
m
decrease and Vmax increase (Manevski et al.,
2
m
(
–
1
–
0
.042 ± 0.002 (CI 95%, 0.037–0.048) to 0.068 ± 0.002 (CI 95%, 0.064–0.073) nmol·min ·mg
1
(
Fig. 2). The results are presented as the average ± S.E., and the calculated 95% CI are given
in parenthesis. Although some deviation from the hyperbolic kinetics was observed at low
concentrations of 1-naphthol, presumably due to technical difficulties in measuring very low
glucuronidation rates, the reaction's kinetics was, generally, well described by the Michaelis-
2
Menten model (r 0.98).
Bisubstrate kinetics of 4-MU glucuronidation by UGT1A9. With the aim of understanding
the enzyme kinetics mechanism of UGT-catalyzed reactions and how it is affected by the
inclusion of BSA, we studied the bisubstrate kinetics of 4-MU glucuronidation by UGT1A9.
The results of this analysis, from the perspective of both substrates, are presented as initial
velocity vs. substrate concentration and as Eadie-Hofstee plots (Fig. 3). The data points were
fitted to eqs. 7–12 (see Materials and Methods) and the best model was selected based on
curve fitting parameters (see Materials and Methods), experimental evidence, and theoretical
considerations (Table 3).

The bisubstrate enzyme kinetics assays, in the absence (as previously done) and in the
presence of BSA, were initially carried out using a lower concentration range of 4-MU, where
substrate inhibition is not clearly apparent to the eye (Fig. 3A and 3B). The primary Eadie-
Hofstee transforms of the data, from the perspective of both substrates, indicated a common
intersection point in the second quadrant, left of the y-axis – a pattern characteristic of ternary-
complex mechanism. Despite many efforts, however, we did not obtain a well-defined
intersection points in Figs. 3A and 3B. Nevertheless, considering the clearly different
intersection point from the one expected in the case of substituted-enzyme mechanism (on the
x-axis in the first quadrant), we think that our results clearly support the formation of a
ternary-complex. The formation of ternary-complex was also supported by the poor fit of the
substituted-enzyme equation (eq. 11) to the experimental data points. On the other hand, the
data (Figs. 3A and 3B) fitted well to the equations for compulsory-order ternary-complex
model based on the steady-state assumption (eq. 7), as well as to the random-order ternary-
complex model based on the rapid-equilibrium assumption (eq. 10). The fit to the compulsory-
order ternary-complex model, based on the rapid-equilibrium assumption (eq. 9), was not as
good, however. The compulsory-order models were also supported by the product and dead-
end inhibition studies (see text below, Figs. 6 and 7, Table 4). The inclusion of BSA did not
qualitatively change the bisubstrate kinetics of 4-MU glucuronidation, even if it significantly
affected the magnitude of the enzyme kinetic parameters (Table 3). Analysis of the bisubstrate
kinetic data with eq. 7 indicates that the inclusion of BSA led to an increase of both the
apparent 4-MU affinity (lower K
m
value) and the reaction limiting velocity (higher Vmax). The
value) in the presence of BSA.
apparent affinity for UDPGA, however, was lower (higher K
m
The second set of bisubstrate kinetics reactions was carried out using higher concentrations of
-MU, in the presence of BSA (Fig. 4). Substrate inhibition became apparent at 4-MU
concentrations higher than 40 µM and, interestingly, was also potentiated by higher
4

concentrations of UDPGA. The data fitted well to the steady-state compulsory-order ternary-
complex model with substrate inhibition (eq. 8, Table 3), yielding a Ksi value for 4-MU of
m
approx. 50-fold higher than the corresponding K value. As in the case of bisubstrate kinetics
at lower concentrations of 4-MU (Fig. 3), the lines in the Eadie-Hofstee plots of the bisubstrate
kinetics with substrate inhibition showed a common intersection point in the second quadrant
(
Figs. 4A and 4B), the pattern indicative of ternary-complex formation. It should be noted,
however, that the intersection pattern appears different from the perspective of UDPGA at
very high concentrations of 4-MU (Fig. 4B, lines marked with dotted line), in the region where
substrate inhibition becomes pronounced. This result suggests that potential substrate
inhibition by the aglycone substrate should always be taken into account when enzyme
kinetics of UDPGA is studied. Otherwise, erroneous conclusions about the enzyme kinetic
parameters may be reached.
In order to understand the overall enzyme kinetic mechanism, we have also investigated the
reverse reaction, an approach that was rarely taken in previous UGT studies, particularly since
recombinant UGTs became available and ensure that the analysis is conducted on a single
enzyme rather than a mixture of different UGTs (Vessey and Zakim, 1972, Rao et al., 1976,
Matern et al., 1991). Hence, the bisubstrate enzyme kinetics, in the presence of BSA, was also
studied for the reverse reaction, namely the formation of 4-MU and UDPGA from 4-MUG and
UDP (Fig. 5). Special attention was paid to the prevention of possible non-enzymatic 4-MUG
hydrolysis and to exclude the possibility that other enzymes in the insect cell membrane are
catalyzing UDPGA formation from 4-MUG and UDP. The latter was examined using the
“Bac-control membranes” that lacked any recombinant UGT and the assays revealed no
significant 4-MU or UDPGA formation upon addition of 4-MUG and UDP. The reverse
reaction, in the presence of BSA, was rather fast under optimal conditions, namely in the
presence of high concentrations of 4-MUG (Fig. 5, Table 3). The results of the bisubstrate

reverse reaction indicated that it follows a ternary-complex mechanism, as can be seen from
the common intersection point in the second quadrant of the Eadie-Hofsetee plots, just left of
the y-axis. It may be noted here that the data points from the reverse reaction are slightly
biphasic and, therefore, may indicate a more complex enzyme kinetic model than currently
applied (eq. 7). Nevertheless, in the current study it was important for us to estimate the
enzyme kinetic parameters of the reverse reaction in the context of the overall enzyme kinetic
mechanism. From this point of view, the equation of compulsory-order ternary-complex
reaction mechanism serves as a good approximation, even if we cannot exclude at this stage a
more complex behavior. Future studies may address this issue in more detail. The K
m
value for
value
4
-MUG was approximately 500-fold higher than the K value for 4-MU, whereas the K
m
m
for UDP was approximately one order of magnitude lower than the corresponding value for
UDPGA. Based on the Haldane relationship for the compulsory-order ternary-complex
mechanism (see Materials and Methods), the thermodynamic equilibrium constant of the
reaction in the presence of BSA is Keq = 574.
One can expect unique relationships between the enzyme kinetic parameters and the individual
rate constants in the compulsory-order ternary-complex mechanism (Cornish-Bowden, 2012).
Unfortunately, due to lack of purified and fully active UGT1A9 enzyme, we could only
determine the relative ratio of the rate constants, not the absolute values. For comparison, the
f
results were normalized by arbitrarily setting the value of V max, the limiting reaction velocity
f
f
in the forward direction, to 1 and then using the expression V max = k cat [E], where [E] is the
molar concentration of the enzyme (Table 5). It is worth noting here that although the first-
order and second-order rate constants cannot be directly compared, a pseudo-first-order rate
constant such as k
1
[AX] can be compared with other first-order constants. These results
f
indicate that the catalytic rate constant in the forward direction, k cat, is about 11-fold higher
r
than the corresponding catalytic rate constant in the reverse reaction k cat
.

Inhibition studies of UGT1A9-catalyzed 4-MU glucuronidation in the presence of 0.1%
BSA. The bisubstrate enzyme kinetic experiments confirmed the existence of ternary-complex.
In order to distinguish between compulsory-order and random-order mechanism, we
performed product inhibition studies with UDP and dead-end inhibition studies with 1-
naphthol. As noted above, 1-naphthol is a high affinity but low turnover substrate for UGT1A9
–1
–1
m
(K = 177 ± 14 nM; Vmax = 0.068 ± 0.002 nmol·min ·mg ) and, under specific experimental
conditions, can be regarded as a 4-MU-competing dead-end inhibitor of UGT1A9.
The results revealed that UDP is a mixed-type and competitive inhibitor of 4-MU
glucuronidation with respect to 4-MU and UDPGA, respectively (Figs. 6A and 7A, Table 4).
On the other hand, 1-naphthol was found to be a mixed-type inhibitor with respect to 4-MU
(
Fig. 6B, Table 4), although with the competitive component of the inhibition clearly
prevailing ( = 7.38 ± 1.75; average ± S.E.; Table 4). Importantly, however, 1-naphthol was
uncompetitive inhibitor with respect to UDPGA (Fig. 7B, Table 4). In addition, it may be
added that the determined values of 1-naphthol’s K
respect to 4-MU (19.8 ± 2.7 nM) are not in good agreement with each other. The observed
discrepancy may be due, at least partially, to the different physical meaning of the K and K
values. While the K is a true equilibrium-dissociation constant of the enzyme•1-naphthol
complex (or in the case of compulsory-order substrate binding, the enzyme•UDPGA•1-
naphthol complex), the K is a pseudo-affinity constant that may or may not equal the true
substrate dissociation constant.
m
(177 ± 14 nM) and mixed-type K
i
with
m
i
i
m

Discussion
We recently found that the “albumin effect” in human UGT1A9 is different than in UGT2B7,
since it both decreases the K and increases the Vmax in the 4-MU and UGT1A9-catalyzed
m
entacapone glucuronidation reactions (Manevski et al., 2011). This finding prompted us to
reexamine the reaction mechanism of UGT1A9 and whether or not it is affected by the
presence of BSA. We selected 4-MU as an aglycone substrate for the detailed enzyme kinetic
analysis since it is a high affinity–high turnover substrate and exhibits substrate inhibition at
high concentrations, a feature typical for many UGT-catalyzed reactions. In addition, 4-MU
and 4-MUG are commercially available and, importantly, are highly fluorescent, making them
suitable for sensitive detection.
For accurate analysis of the BSA effect it is essential to determine the free drug concentration
in the presence of the used BSA concentration. For such measurements we have now used the
RED method (Waters et al., 2008) and compared the results with our previous data that were
obtained using ultrafiltration (Manevski et al., 2011). The two methods provide very similar
results for both entacapone and 4-MU binding to BSA, when taking into account the non-
2
specific drug binding to the device (Fig. 1). The inclusion of MgCl , UDPGA, or UDP did not
significantly alter the substrates binding to BSA. In contrast, the addition of membrane
samples (0.02–0.2 mg/mL) significantly modulated the entacapone binding to 0.1% BSA and
should be taken into account (Manevski et al., 2011).
To examine whether or not BSA affects the reaction mechanism of UGT1A9 we have used
bisubstrate kinetic analysis of 4-MU glucuronidation by UGT1A9. The results, both in the
forward and the reverse directions, strongly suggested that the 4-MU glucuronidation reaction
proceeds through the formation of ternary-complex, regardless of whether or not BSA was
added to the assay (Figs. 3, 4, and 5; Table 3). The bisubstrate kinetic data were best described

by a steady-state compulsory-order ternary-complex model, even though evidence from
complementary inhibition experiments may be needed to fully prove this (see below). BSA
presence affected the forward reaction by lowering the K
m
value for 4-MU and increasing the
value of UGT1A9 for
V
max value. In addition, and somewhat surprisingly, it increased the K
m
UDPGA (Fig. 3, Table 3). The inclusion of BSA, however, did not qualitatively affect the
bisubstrate kinetics model (Fig 3, see Eadie-Hofstee insets).
The analysis of bisubstrate kinetics results in the forward and reverse directions, when both
were assayed in the presence of BSA, revealed that UGT1A9 has about 500-fold higher
affinity for 4-MU than for 4-MUG (Table 3). Interestingly, the apparent affinity of the enzyme
for UDP, as measured in the reverse reaction, is an order of magnitude higher than for
UDPGA in the forward reaction (Table 3). Taken together, the results suggest that the reverse
reaction is mainly limited by the poor affinity of the enzyme for 4-MUG. Another factor that
contributes to making the reaction proceed almost exclusively in the forward direction in the
human cell is that the k–2 (first-order rate constant for E•AX•B
E•AX + B reaction) is much
smaller than k
3
(first-order rate constant for E•AX•B
E•A + BX reaction) (Fig. 8 and Table
5
). A comparison of the relative individual rate constants also shows that the first-order rate
constants in the forward direction, such as k
3
and k
4
, are within the same order of magnitude as
k
–1, an observation that supports the steady-state assumption (Fig. 8, Table 5). Based on the
measured enzyme kinetic parameters and the Haldane relationship, the equilibrium constant of
the overall reaction is large, Keq = 574. Nevertheless, as we clearly show in this study (Fig. 5),
the reverse reaction can also be performed and analyzed under, more-or-less, conditions of
standard in vitro UGTs assays.
The presence of substrate inhibition at high concentrations of 4-MU (Fig. 4), that appears to be
stimulated by the presence of high UDPGA concentrations, provides evidence for a steady-

state compulsory-order ternary-complex reaction mechanism (see also Luukkanen et al.,
005). In this mechanism, either the second substrate or the first product of the reaction may
2
bind to a "wrong" binary complex. The results of the bisubstrate kinetic analyses support the
assumption that in the case of the studied reaction, UDPGA and 4-MU are the first and second
binding substrates, respectively, and that substrate inhibition probably occurs due to 4-MU
binding to a “wrong” binary complex, E•UDP, rather than to the “correct” binary complex,
E•UDPGA (Fig. 8). The measured relatively high affinity of UDP to the free enzyme may
explain why substrate inhibition is commonly observed in UGT-catalyzed reactions, especially
if the initial rate conditions are not strictly followed and the UDP concentration increases due
to its accumulation during the forward reaction (Table 3). This should raise awareness about
potential problems in substrate depletion experiments with the UGTs. In such experiments,
contrary to initial-rate assays, the accumulation of UDP is likely to slow down the aglycone
substrate depletion.
Substrate inhibition can also occur in the random-order ternary-complex mechanism, where 4-
MU may also bind to E•UDP complex. However, if the rapid-equilibrium assumption is valid,
the E•UDP complex concentration is zero in the absence of accumulated or added products.
Since 4-MU cannot bind to enzyme species that are not present, the rapid-equilibrium random-
order ternary-complex mechanism can be excluded based on this observation. However, the
non-rapid-equilibrium random-order ternary-complex mechanism may not be ruled out based
on bisubstrate kinetics only. The rapid-equilibrium compulsory-order ternary-complex,
however, may be excluded based on herein measured and reported rate constant magnitudes
(
Table 5; Luukkanen et al., 2005), substrate inhibition, and poor fit to experimental data.
UDP was found to be a competitive inhibitor with respect to UDPGA, and mixed-type
inhibitor with respect to 4-MU (Figs. 6A and 7A, Table 4). Such inhibition patterns agree well

with the previously published data (Luukkanen et al., 2005, Fujiwara et al., 2008) and are
possible for both random-order and compulsory-order ternary-complex mechanisms. However,
if the reaction follows the compulsory-order ternary-complex mechanism, as indicated by the
bisubstrate kinetic analyses and substrate inhibition, the UDP inhibition results support the
suggestion that UDPGA is the first binding substrate in a compulsory-order mechanism.
In line with previous finding in the absence of BSA (Luukkanen et al., 2005), 1-naphthol is a
predominantly competitive inhibitor of UGT1A9 with respect to 4-MU ( = 7.38 ± 1.75) but,
importantly, uncompetitive with respect to UDPGA (Figs. 6B and 7B, Table 4). This result
indicates that 1-naphthol, an inhibitor that probably competes with 4-MU for the aglycone
substrate-binding site, does not compete for the same enzyme species as UDPGA. The
uncompetitive inhibition pattern arises from 1-naphthol binding to the pre-formed E•UDPGA
complex, rather than to the free enzyme. This uncompetitive inhibition provides strong
evidence that UGT substrates are binding in a compulsory fashion in which the initial binding
of UDPGA increases the affinity for the aglycone substrate. Such an affinity increase could be
a result of a conformational change in the enzyme upon UDPGA binding, the involvement of
the bound UDPGA molecule itself in the binding of the aglycone substrate (see Fig. 9 o f
Itaaho et al., 2010), or both. We can also expect that the reverse reaction should be structurally
analogous to the forward reaction, and that the second product of the forward reaction (UDP)
should be the structural analogue of the first substrate (UDPGA).
Based on the available evidence, UGT1A9 follows a steady-state compulsory-order ternary-
complex mechanism, regardless of whether or not BSA is present (Fig. 8). One may then ask
how BSA interferes with this reaction mechanism to affect the apparent substrate affinities and
the reaction Vmax. Taking into account that the presence of BSA increases the apparent affinity
for 4-MU and decreases the apparent affinity for UDPGA, it m ay be proposed that BSA

removes internal inhibitors that are competitive and/or noncompetitive with respect to the
aglycone substrate, but are uncompetitive with respect to UDPGA. The uncompetitiveness of
the BSA-removed inhibitors with respect to UDPGA would explain why the affinity for this
cosubstrate apparently decreases in the presence of BSA. Such inhibitors, tentatively marked
as I
1
and I in the reaction scheme (Fig. 8) would not (or poorly) bind to the free enzyme, but
2
would bind with higher affinity to the binary E•UDPGA complex or the ternary E•UDPGA•4-
MU complex.
If the UGT1A9 inhibitor(s) source is the membrane in which the enzyme is located, it may be
suggested that the differences in lipid composition between the insect cell membranes (see
Marheineke et al., 1998, for the lipid composition of Sf9 membranes) to the human liver
membranes could lead to differences in the BSA effects between the recombinant UGT and
the native enzyme. Presently, we cannot discard this possibility, particularly regarding the
magnitude of the BSA effect when different substrates are used. Nevertheless, recent studies
on the BSA effects demonstrated only minor influences of the membrane source on the BSA
effects in UGT1A9 (Manevski et al., 2011; Shiraga et al., 2012), or UGTs 1A1, 1A4, 1A6, and
2
B7 (Walsky et al., 2012). Hence, the currently available results do not indicate significant
differences between Sf9 membranes and HLM as sources of the inhibitory fatty acids that are
removed by BSA, when present during the glucuronidation reaction. While the exact nature
and number of these inhibitors is currently unknown, the improved understanding of the UGT
reaction mechanism and the BSA effects may help to rationalize and predict the BSA effect in
future.

Acknowledgements
We would like to thank Johanna Mosorin for skilful technical assistance.
Author contributions:
Participated in research design: N.M., M.F.
Conducted experiments: N.M.
Contributed new reagents or analytical tools:
Performed data analysis: N.M.
Wrote or contributed to the writing of the manuscript: N.M., J.Y-K., M.F.

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