Susan G. Cady2 and Masanori Sono: Archives of Biochemistry AND Biophysics
Susan G. Cady2 and Masanori Sono: Archives of Biochemistry AND Biophysics
Susan G. Cady2 and Masanori Sono: Archives of Biochemistry AND Biophysics
1 -Methyl-DL-tryptophan, P-(3-Benzofuranyl)-DL-alanine
(the Oxygen Analog of Tryptophan), and ,&[3-
Benzo(b)thienyl]-kalanine (the Sulfur Analog
of Tryptophan) Are Competitive Inhibitors
for lndoleamine 2,3-Dioxygenase’
326 0003.9861/91$3.00
Copyright 0 1991 by Academic Press, Inc.
All rights of reproduction in any form reserved.
Trp ANALOG COMPETITIVE INHIBITORS FOR INDOLEAMINE 2,3-DIOXYGENASE 327
(ll), while this compound is a substrate for the rabbit Enzymes. Indoleamine 2,3dioxygenase was purified from rabbit
intestinal (12,13) and human placental dioxygenases (14). small intestine by the method of Shimixu et al. (13) except that the final
isoelectrofocusing step was omitted and instead step 6 (Sephadex G-
On the other hand, many inhibitors have been known
100 chromatography) was repeated 2-4 times. The purified native ferric
for tryptophan 2,3dioxygenase, another heme-containing enzyme exhibited an AM/Am value of 1.7-1.8 in 20 mu potassium
dioxygenase (1516) that catalyzes the same reaction as, phosphate buffer at pH 6.0 and 25’C and was >70% pure as judged by
but is distinct from, indoleamine 2,3-dioxygenase. In ad- sodium dodecyl sulfate gel electrophoresis (4). The amount of the enzyme
dition to the above-mentioned compounds (except for 4- was expressed in terms of its heme content based on the absorbance at
405 nm [c = 159 mM-’ cm-’ at pH 6.0 and 25°C (S)]. Bovine liver catalase
phenylimidazole that has not been examined), various was a product of Sigma.
tryptophan analogs and derivatives such as &hydroxy-L-
Enzyme kinetic experiments. The assays of indoleamine 2,3dioxy-
Trp, tryptamine, serotonin (&hydroxytryptamine), 5 genase were performed at 25°C using 50 nM dioxygenase, either D- or
methyl-Trp, and 5-fluoro-Trp, all of which are substrates L-Trp as a substrate, and an ascorbic acid-methylene blue (+catalase)
for indoleamine 2,3dioxygenase (13, 17), have been re- cofactor system as described previously for the standard assay (3) by
ported to inhibit tryptophan 2,3dioxygenase in either a continuously following the absorbance increase at 321 nm [c = 3.75
mM-’ cm-’ (21)] due to the formation of N’-formylkynurenine in the
competitive or a noncompetitive [an inhibitor binds absence and presence of the Trp analog inhibitors. Enzyme kinetic data
equivalently to both the free (E) and the substrate-bound [the initial rate of the product formation (V) vs substrate (Trp) con-
(ES) enzyme] manner with respect to the substrate L- centration] were analyzed by employing three types of plots, the Line-
Trp (15,18, 19). weaver-Burk (or the double-reciprocal) plot (l/Vvs l/[substrate]), the
Eadie-Hofstee plot (V vs V/[substrate]), and the Hanes-Woolf plot
Inhibitors for these two dioxygenases, especially sub- ([substrate]/Vvs [substrate]) (22).
strate analog competitive inhibitors, may serve as useful
Preparations of indoleamine $3-dioxygenuse derivatives and their ti-
probes for understanding what physical and electronic trations with Trp analogs and heme &an&. Direct or indirect (com-
structural factors are required for an organic compound petitive) titration experiments for the binding of L-Trp, Trp analogs,
not only to be able to bind to the catalytic site of the and the heme ligands azide and I-phenylimidasole to the ferric and the
enzyme but also, more importantly, to serve as a substrate ferrous dioxygenases were carried out in 0.1 M potassium phosphate
buffer at 25°C with optical absorption and CD spectroscopy as described
for the enzyme. Such inhibitors might thus provide a clue previously (3,9). Analysis of titration data has been described elsewhere
to the understanding of the mechanism of action of these (3, 23). Due to the relatively low solubilities, the Trp analogs used in
enzymes which has not yet been clarified. this study were added from their 5-6 mM stock solutions in water or in
In the course of a study on the effects of Trp modifi- -5 mM HCl (cf. 50 mM D- and L-Trp stock solutions in water). Ferrous
derivatives (the unligated form and its complexes with CO and NO) of
cations on the catalytic activity of indoleamine 2,3diox-
the dioxygenase were prepared as described previously (3,4).
ygenase (17), we have discovered three Trp analogs which
Spectroscopic measurements. Optical absorption spectra and CD
serve as potent competitive inhibitors with respect to D- spectra were recorded on a Varian Cary 219 spectrophotometer and a
Trp and L-Trp for the rabbit intestinal dioxygenase. These Jasco J-500A spectropolarimeter, respectively. Both instruments were
compounds are 1-methyl-DL-Trp, @-(3-benzofuranyl)-DL- equipped with a circulator for temperature control (+l”C). All experi-
alanine (the oxygen analog of Trp), and B- [3- ments were carried out at 25’C with a O.l- or 0.2-cm cuvette. Except
for updated instrumentation (Jasco J-500A spectropolarimeter and its
benzo(b)thienyl] -DL-alanine (the sulfur analog of Trp) data acquisition and storage systems and the use of IBM personal com-
each of which has a substitution at the indole nitrogen puters), the basic methods and instrumental conditions used have been
atom (see Fig. 1 for their structures). Enzyme kinetic described elsewhere (23).
studies of the inhibition mechanism and spectroscopic
equilibrium binding studies of the interactions of these
Trp analogs with the dioxygenase will be reported in this RESULTS
paper.
Among the various Trp analogs examined, including
EXPERIMENTAL PROCEDURES those having a substitution of a fluorine atom or a methyl
group for a single hydrogen atom on the indole ring, and
Materials. L-Trp was purchased from Sigma. D-Trp and l-methyl-
DL-T~~ were purchased from Aldrich. @-[3-Benzo(b)thienyl]-DL-alanine those having a heteroatom substitution of a sulfur or OX-
hydrochloride, the sulfur analog of Trp, was a gift from Professor Tal- ygen atom for the indole nitrogen atom (17), the following
mage R. Bosin at Indiana University. @-(3-Benzofuranyl)-DL-alanine, three compounds were found to markedly inhibit the cat-
the oxygen analog of Trp, was a gift from the National Institute of alytic activity of indoleamine 2,3dioxygenase with D-Tip
Mental Health Chemical Synthesis Program (c/o Dr. J. Stephen Ken- and L-Trp as substrates: 1-methyl-DL-Trp, P-(3-benzo-
nedy and Dr. Albert A. Manian, NIMH). All of these Trp analogs were
an approximately 1:l mixture of the DL-forms as examined with reverse furanyl)-DL-alanine (the oxygen analog of Trp), and p-
phase high performance liquid chromatography (Cyclobond III chiral [3-benzo(b)thienyl] -DL-alanine (the sulfur analog of Trp)
column, ASTEC Inc., Whippang, New Jersey) (20). The sulfur and the (see Fig. 1). None of these Trp analogs were found to
oxygen analogs were free of DL-Trp. Since the commercial samples of serve as substrates for the dioxygenase in this study.
1-methyl-DL-Trp contained -4% DL-Trp (17), it was recrystallized from Figure 2 shows a typical double-reciprocal plot for the
hot water to decrease the DL-Trp contamination to about 2% (1% for
each isomer). The DL mixtures of these three Trp analogs were used in conversion of D-Trp to N’-formylkynurenine by the diox-
this study. ygenase at pH 8.0 in the absence and presence of various
328 CADY AND SON0
coo- B
/
Wavelength (nm>
TABLE III
Effects of Indole 1-N-Substituted TRP Analogs on the Dissociation Constant (I&) of the Ferric Indoleamine 2,3-Dioxygenase
(IDO)-Azide Complex and the Ferrous IDO-4-Phenylimidazole (4-PheImid) Complex”
Trp analog Concn. of Trp Kd of ferric IDO-azide Concn. of Trp Kd of ferrous IDO-4-PheImid
added* analog (mM) complex (mM) analog (mM) complex (mM)
’ All values were determined in 0.1 M potassium phosphate buffer, pH 7.0, and at 25’C with optical absorption spectroscopy.
* See footnote b to Table I for the abbreviation and chemical names of the compounds. L-Trp is included for comparison.
332 CADY AND SON0
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