Inhibitory substrate binding site of human indoleamine 2,3-dioxygenase

Article abstract of DOI:10.1021/ja9029768

(Graph Presented) Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway. Due to its immunosuppressive function, hIDO has been recognized as an important drug target for cancer. Here we report evidence supporting the presence of an inhibitory substrate binding site (S_i) in hIDO that is capable of binding molecules with a wide variety of structures, including substrates (L-Trp and 1-methyl-L-tryptophan), an effector (3-indole ethanol), and an uncompetitive inhibitor (Mitomycin C). The data offer useful guidelines for future development of more potent hIDO inhibitors; they also call for the re-evaluation of the action mechanism of Mitomycin C (MtoC), a widely used antitumor chemotherapeutic agent.

Full text of DOI:10.1021/ja9029768

Published on Web 08/20/2009
Inhibitory Substrate Binding Site of Human Indoleamine 2,3-Dioxygenase
Changyuan Lu, Yu Lin, and Syun-Ru Yeh*
Department of Physiology and Biophysics, Albert Einstein College of Medicine,
1300 Morris Park AVenue, Bronx, New York 10461
Received April 14, 2009; E-mail: syeh@aecom.yu.edu
Indoleamine 2,3-dioxygenase (IDO) is a nonhepatic intracellular
heme-containing enzyme.1 It catalyzes the conversion of L-
tryptophan (L-Trp) to N-formylkynurenine (NFK), the initial and
rate-determining step of the kynurenine pathway, by inserting both
atoms of dioxygen across the C2dC3 bond of the indole moiety of
L-Trp.2 IDO was first discovered by Hayaishi et al. more than four
decades ago.3 Since then, its structural and functional properties
were extensively studied until the early 1990s. This field of research
was invigorated recently due to the discoveries that IDO is linked
to a variety of immune-related pathophysiological conditions and
that IDO is a potential target for pharmacological intervention
against cancer.4 Here we report evidence supporting the presence
of an inhibitory substrate binding site (Si site) in human IDO
(hIDO), which is capable of binding substrates (L-Trp and 1-methy-
L-tryptophan), an effector (3-indole ethanol), and an uncompetitive
inhibitor (Mitomycin C). The structures of these molecules are
illustrated in Scheme 1.
Figure 1. Michaelis-Menten plots of the dioxygenase reaction of hIDO
(91nM) at pH 7.4 with respect to [L-Trp] (a) and [D-Trp] (b) under air-
saturated conditions. The inset in (b) shows the corresponding plot for the
reaction of hIDO with 36 µM L-Trp with respect to [O2] at pH 7.4.
V ) Vmax × [S]/(Km + [S] × (1 + [S]/Ksi))
(1)
Here, [S] is the substrate concentration; Vmax and Km are
Michaelis-Menten constants. The best-fitted parameters are listed
in Table 1. The data indicate that, under steady-state conditions,
the probability of L-Trp binding to the ferric enzyme, followed by
its reduction to the active ferrous state, is negligible (similar to
that concluded by Sono et al. for rIDO5), as the L-Trp dissociation
constant of the ferric enzyme (Kd ) 0.9 mM) is much higher than
the Km (15 µM) and Ksi (0.17 mM). It is noteworthy that the
physiological concentration of L-Trp, ∼50-100 µM,9 lies right
between the observed Km and Ksi values.
Scheme 1. Molecular Structures of 3-Indole Ethanol (IDE),
Mitomycin C (MtoC), and 1-Methyl-tryptophan (1MTrp)
Table 1. Michaelis-Menten and Inhibition Constants of hIDO
Associated with Its Reaction with L-Trp in the Presence and
Absence of IDE and Those Associated with D-Trp and L-1MTrp
(kcat Values Calculated from Vmax/[hIDO])
The activity of hIDO was first examined as a function of L-Trp
concentration, following the mixing of the ferric enzyme with a
methylene blue-ascorbate reducing system.3 As shown in Figure
1a, the activity of hIDO follows typical Michaelis-Menten behavior
at [L-Trp] < 50 µM; further increase in [L-Trp] caused a decrease
in the activity, signifying substrate-inhibition behavior, well-known
for rabbit IDO (rIDO).3,5 It was generally believed that, at high
[L-Trp], the substrate can bind to the ferric enzyme, thereby
retarding the turnover of the enzyme by inhibiting its reduction to
the active ferrous state.5 This scenario, however, can be excluded
on the basis of two new observations: (1) the dissociation constant
of L-Trp for the ferric IDO (Kd ) 0.9 mM; see Figure S1) is
significantly higher than the self-inhibition constant, Ksi (0.17 mM,
Vide infra), and (2) the redox potential of the L-Trp-bound ferric
hIDO is ∼46 mV higher than that of the substrate-free enzyme,6
indicating L-Trp binding to the ferric enzyme does not prevent its
reduction.
We propose that the substrate-inhibition behavior of hIDO is a result
of the binding of a second L-Trp in an inhibitory Si site of the enzyme.
This hypothesis is consistent with recent computational data showing that
the distal pocket of hIDO is flexible enough to accommodate both L-Trp
and an indole derivative.7 On the basis of this scenario, the data shown in
Figure 1a were fitted with the following equation8 by assuming L-Trp
can bind to the active site, as well as the Si site.
L-Trp
+IDE2.5mM +IDE5.1mM
D-Trp
L-1MTrp
kcat (s-1
Km (µM) 15 ( 2
)
3.1 ( 0.2 3.2 ( 0.4 3.5 ( 0.6 5.9 ( 0.3
0.064 ( 0.003
62 ( 9
5.0 ( 0.8
16 ( 3
15 ( 3 (2.6 ( 0.2) × 103
Ksi (mM) 0.17 ( 0.2 0.48 ( 0.05 1.0 ( 0.1
-
Similar activity studies were carried out with D-Trp. No
substrate inhibition was observed (Figure 1b), indicating substrate
inhibition is stereospecific to the L isomer, as reported for rIDO.3
The Michaelis-Menten fit of the data shows, as compared to
the L-Trp reaction, the kcat is 2-fold faster (5.9 s-1), while the
Km value is 170-fold larger (2.6 mM). The data demonstrate that
the substrate stereoselectivity of hIDO is a result of its
preferential binding of the L-isomer. Additional activity studies
were conducted as a function of [O2] in the presence of 36 µM
L-Trp (see the insert in Figure 1b). The kcat value was determined
to be 2.9 s-1, similar to that determined by the data shown in
Figure 1a. Km for O2 was determined to be 42 µM, which is
near the physiological level of oxygen, ∼50-76 µM,10 and is
comparable to that of an analogous enzyme, tryptophan 2,3-
dioxygenase, (40 µM)11 and those of monooxygenases, such as
NOSs (6-24 µM)12 or P450s (1-15 µM).13
IDE has been long recognized as an effector for rIDO.14 It was
believed IDE enhances the activity of rIDO by improving its kcat
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C O M M U N I C A T I O N S
via binding to an accessory binding site located near the catalytic
site of IDO.14 To investigate if the IDE binding site coincides with
the aforementioned Si site, we examined the hIDO activity in the
presence of 2.5 and 5.1 mM IDE. As shown in Figure 2a, the
presence of IDE led to higher activity and less pronounced substrate
inhibition. The best fit of data with eq 1 indicates IDE does not
affect kcat and Km but causes the elevation of Ksi from 0.17 to 0.48/
1.0 mM (for 2.5/5.1 mM IDE, see Table 1), indicating IDE
competes with L-Trp for the Si site. As IDE bound to the Si site,
unlike L-Trp, does not retard the active site catalysis, the apparent
activity is higher than that in its absence. The data clearly
demonstrate that IDE acts as an effector by binding to the Si site,
thereby blocking it from L-Trp binding, instead of by facilitating
kcat, as generally believed.
MtoC, a quinone-containing natural product from Streptomyces,
has been widely used as a chemotherapy drug for several types of
cancer.15 It is believed that the antitumor activity of MtoC is a
result of its ability to generate oxygen radicals upon reduction,
thereby inhibiting DNA synthesis. The recent findings that hIDO
is a potential therapeutic target for cancer treatment and that quinone
is a potent pharmacophore for hIDO inhibition16-18 prompted us
to investigate if hIDO could function as a pharmacological target
of MtoC. We examined the hIDO activity as a function of MtoC
concentration. The Lineweaver-Burk plot of the data (Figure 2b)
shows MtoC indeed inhibits hIDO in an uncompetitive fashion with
an inhibition constant (Ki) of ∼25 µM. The data indicate that, as
an uncompetitive inhibitor, MtoC binds only to the L-Trp-bound
enzyme, not the substrate-free enzyme. We propose that L-Trp
binding in the active site induces structural changes to the Si site
to accommodate the MtoC molecule and that the occupancy of the
Si site by MtoC inhibits the active site catalysis.
Figure 2. Michaelis-Menten plots of the hIDO reaction at pH 7.4, in the
absence or presence 2.5 or 5.1 mM IDE (a) and the Lineweaver-Burk
plots of the steady-state activities of hIDO in the presence of various
concentrations of MtoC (b) and L-1MTrp (c).
Recently, hIDO has emerged as a therapeutic target for cancer,4
leading to an active search for potent inhibitors. Our data show
MtoC effectively inhibits hIDO, calling for re-evaluation of the
action mechanism of this commonly used antitumor chemothera-
peutic agent. They also suggest the newly discovered quinone-
containing hIDO inhibitors,16-18 like MtoC, may selectively bind
to the Si site. Taken together the data presented in this work provide
the first glimpse of the Si site that offers potential guidelines for
future development of more efficient hIDO inhibitors.
Acknowledgment. We would like to thank Dr. Denis L.
Rousseau for valuable discussions.
Supporting Information Available: The Materials and Methods, the
absorption spectra of the substrate-free and L-Trp-bound ferric hIDO and the
associated L-Trp titration data, as well as the Michaelis-Menten plot with
respect to L-1MTrp and a cartoon illustrating the Si site of hIDO. This material
is available free of charge via the Internet at http://pubs.acs.org.
As a control experiment, the inhibitory effect of 1MTrp was
examined. 1MTrp has been widely used as an hIDO inhibitor.19
As shown in Figure 2c, the L-isomer acts as a competitive inhibitor,
with a Ki of 32 µM, while the D-isomer exhibits no inhibitory effect
(data not shown), consistent with that reported by Hou et al.20
Additional studies show, in the absence of L-Trp, L-1MTrp itself
can act as a substrate, although kcat is 50-fold lower than that of
L-Trp (Figure S2), similar to that reported by Chauhan et al.21 In
addition, our data show that L-1MTrp exhibits substrate-inhibition
behavior, indicating, like L-Trp, L-1MTrp binds to the Si site at
elevated concentrations, thereby inhibiting hIDO activity. The 29-
fold higher Ksi value, as compared to that of L-Trp (Table 1),
indicates the bulky methyl group on the indole nitrogen significantly
lowers its affinity toward the Si site.
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substrate binding sites, an active binding site and a Si site (as
illustrated in a cartoon shown in Figure S3). The binding of L-Trp
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