Transition-state stabilization by a mammalian reductive dehalogenase

Full text of DOI:10.1021/ja990693n

4722
J. Am. Chem. Soc. 1999, 121, 4722-4723
Scheme 1
Transition-State Stabilization by a Mammalian
Reductive Dehalogenase
Munetaka Kunishima,† Jessica E. Friedman, and
Steven E. Rokita*
Department of Chemistry and Biochemistry
UniVersity of Maryland College Park, Maryland 20742
Scheme 2
ReceiVed March 3, 1999
Enzyme-catalyzed dehalogenation is typically associated with
xenobiotic metabolism and in particular with microorganisms that
detoxify environmental pollutants.1,2 Both aliphatic and aromatic
substrates are susceptible to dehalogenation through mechanisms
that include oxidation, reduction, and hydrolysis. Oxidative
processes are usually associated with aerobic organisms, whereas
reductive processes are usually associated with anaerobic organ-
isms. Remarkably, mammals have the additional ability to
promote reductive deiodination of the hormone thyroxine (3-[4-
(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenylalanine), its me-
tabolites, and related intermediates including iodotyrosine. A series
of selenoenzymes found in tissues such as brown fat, liver, kidney,
and the central nervous system are responsible for the reduction
and deiodination of thyroxine and the concomitant oxidation of
glutathione.3 In contrast, an iodide salvage enzyme in the thyroid
mediates reduction and deiodination of iodo- and diiodotyrosine
with consumption of NADPH (Scheme 1).4 Little mechanistic
data has yet to be gathered on these mammalian reactions, and
we now report compelling evidence for a key intermediate
proposed in catalysis of iodotyrosine deiodinase.
Both direct aromatic substitution and halophilic reaction of a
nonaromatic tautomer (see below) can be considered for this
reductive process. However, aromatic substitution via nucleophilic
addition and formation of an anionic Meisenheimer intermediate
(SNAR) is disfavored for electron-rich targets. The weak C-I bond
might instead suggest a related homolytic process (SRN1), and
yet model conditions necessary for such a dehalogenation remain
far from physiological.5 Preliminary studies have also suggested
that diiodotyrosine is stable to one-electron reductants including
sulfite, metabisulfite, ferrocyanide, and dithionite.4a Electron-rich
aryl halides are most commonly dehalogenated via Lewis acid-
catalyzed two-electron processes.6 Tautomerization (or possibly
protonation) to form the nonaromatic intermediate illustrated in
Scheme 2 facilitates halophilic attack and cleavage of the C-I
bond. This type of process has been proposed for triphenylphos-
phine-dependent debromination of o-bromophenol and bromo-
aniline,7 AlCl3- and GaCl3-catalyzed deiodination of diiodocresol,8
hydriodic acid-dependent deiodination of aryl iodides,9 and most
recently, biomimetic coupling of two diiodotyrosines.10 Similar
mechanisms have been proposed for the biological deiodination
of thyroxine by a selenocysteinyl residue11 and dechlorination of
tetrachlorohydroquinone by a cysteinyl residue or glutathione.12
The active site of iodotyrosine deiodinase has not yet been
characterized but may also include a cysteinyl residue.13 The
ability of this enzyme to stabilize the proposed16 nonaromatic
tautomer is described below by the binding affinity of pyridonyl-
containing mimics of this intermediate.
The initial targets, D,L-3-(2-pyridon-5-yl)alanine 1 and D,L-3-
(N-methyl-2-pyridon-5-yl)alanine 2, were constructed using a
standard condensation with diketopiperazine (Scheme 3).17 The
necessary aldehyde was prepared by sequential bromination,
lithiation, and finally formylation of 2-methoxypyridine (see
Supporting information for experimental details).18 The intermedi-
ate diketopiperazine was reduced and hydrolyzed by HI and
(7) Hoffmann, H.; Michael, D. Chem. Ber. 1962, 95, 528-535
(8) Tashiro, M.; Makishima. T.; Horie, S. J. Chem. Res. 1987, 342-343.
(9) (a) Gold, V.; Whittaker, M. J. Chem. Soc. 1951, 1184-1190. (b)
Choguill, H.; Ridd, J. J. Chem. Soc. 1961, 822-826. (c) Norman, R. O. C.;
Taylor, R. Electrophilic Substitution in Benzenoid Compounds; Elsevier: New
York, 1965; pp 247-249.
(10) Bell, N. V.; Bowman, W. R.; Coe, P. F.; Turner, A. T.; Whybrow, D.
Tetrahedron Lett. 1997, 38, 2581-2584.
(11) Berry, M. J.; Kieffer, J. D.; Harney, J. W.; Larsen, P. R. J. Biol. Chem.
1991, 266, 14155-14158. (b) Sun, B. C.; Harney, J. W.; Berry, M. J.; Larsen,
P. R. Endocrinology 1997, 138, 5452-5458. (c) Vasil’ev, A. A.; Engman, L.
J. Org. Chem. 1998, 63, 3911-3917.
(12) (a) Willett, W. S.; Copley, S. D. Chem. Biol. 1996, 3, 851-857. (b)
McCarthy, D. L.; Navarrete, S.; Willett, W. S.; Babbitt, P. C.; Copley, S. D.
Biochemistry 1996, 35, 14634-14642. (c) McCarthy, M. L.; Louie, D. F.;
Copley, S. D. J. Am. Chem. Soc. 1997, 119, 11337-11338.
(13) Thiols exhibit a unique affinity for aryl and aliphatic iodo substitu-
ents.14,15 For example, chloro- and bromomethyl ketones react with thiols to
generate a mixture of substitution and reduction products, whereas iodomethyl
ketones react under equivalent conditions to generate the reduced methyl ketone
exclusively.15
(14) (a) Montanari, S.; Paradisi, C.; Scorrano, G. J. Org. Chem. 1993, 58,
5628-5631. (b) Patai, S., Ed. The Chemistry of the Carbon-Halogen Bond;
Wiley: New York, 1973; Vols. 1 and 2.
(15) Seshadri, R.; Pegg, W. J.; Israel, M. J. Org. Chem. 1981, 46, 2596-
2598.
(16) Hartmann, K.; Hartmann, N. Z. Chem. 1971, 11, 344-345.
(17) Niemann, C.; Lewis, R. N.; Hays, J. T. J. Am. Chem. Soc. 1942, 64,
1678-1682.
(18) (a) Spinner, E.; White, J. C. B. J. Chem. Soc. (B) 1966, 991-995. (b)
Bargar, T. M.; Wilson, T.; Daniel, J. K. J. Heterocycl. Chem. 1985, 22, 1583-
1592. (c) Comins, D. L.; Killpack, M. O. J. Org. Chem. 1990, 55, 5, 69-73.
† Permanent address: Faculty of Pharmaceutical Sciences, Kobe Gakuin
University, Nishi-ku, Kobe 651-2180, Japan.
(1) (a) Kirk, K. L. Biochemistry of the Halogenated Organic Compounds;
Plenum Press: New York, 1991. (b) Fetzner, S.; Lingens, F. Microbiol. ReV.
1994, 641-685. (c) Copley, S. D. Chem. Biol. 1997, 4, 169-174.
(2) (a) Abramowicz, D. A. Crit. ReV. Biotechnol. 1990, 10, 214-251. (b)
Wackett, L. P.; Schanke, C. A. In Metals in Biological Systems; Sigel, H.,
Sigel, A., Eds.; Marcel Dekker: New York, 1992; Vol. 28, pp 329-356. (c)
Neumann, A.; Wohlfarth, F.; Diekert, G. J. Biol. Chem. 1996, 271, 16515-
16519. (d) Romanov, V.; Hausinger, R. P. J. Bacteriol. 1996, 178, 2656-
2661. (e) Maymo´-Gatell, X.; Chien, Y.-T.; Gossett, J. M.; Zinder, S. H. Science
1997, 276, 1568-1571. (f) Quense, J. F.; Mueller, S. A.; Jain, M. K.; Tiedje,
J. M. Science 1998, 280, 722-724.
(3) (a) Berry, M. J.; Banu, L.; Larsen, P. R. Nature 1991, 349, 438-440.
(b) Larsen, P. R. Biochem. Soc. Trans. 1997, 25, 588-592.
(4) (a) Goswami, A.; Rosenberg, I. N. Endocrinology 1977, 101, 331-
341. (b) Rosenberg, I. N.; Goswami, A. J. Biol. Chem. 1979, 254, 12318-
12325. (c) Goswami, A.; Rosenberg, I. N. J. Biol. Chem. 1979, 254, 12326-
12330.
(5) (a) Alam, N.; Amotore, C.; Combellas, C.; Pinson, J.; Save´ant, J.-M.;
Thie´bault, A.; Verpeaux, J.-N. J. Org. Chem. 1988, 53, 1496-1504. (b)
Moreno, M.; Gallardo, I.; Bertra´n, J. J. Chem. Soc., Perkin Trans. 2 1989,
2017-2021. (c) Bunnett, J. F. Acc. Chem. Res. 1992, 25, 2-9. (d) Das, T. N.
J. Phys. Chem. 1998, 102, 426-433.
(6) (a) de la Mare, P. B. D.; Swedlund, B. E. in Chemistry of the Carbon-
Halogen Bond; Patai, S., Ed.; Wiley: New York, 1973; Chapter 7, pp 407-
548. (b) Zefirov, N. S.; Mahkon’kov, D. I. Chem. ReV. 1982, 82, 615-624.
10.1021/ja990693n CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/29/1999

Communications to the Editor
J. Am. Chem. Soc., Vol. 121, No. 19, 1999 4723
Table 1. Summary of Kinetic and Binding Constants for
Scheme 3
Iodotyrosine Deiodinasea
Scheme 4
a All inhibitors bound competitively with respect to diiodotyrosine,
and hence, KI values were calculated accordingly from their concentra-
tion-dependent effect on the apparent Km of diiodotyrosine.
efficient but unremarkable inhibitor with a KI that was ap-
proximately 9-fold lower than the Km for diiodotyrosine (Table
1). However, methylation of the pyridonyl ring generated an
analogue 2 with a highly significant 46-fold increase in enzyme
affinity relative to 1. The KI of this N-methyl derivative is thus
more than 380-fold lower than the Km of diiodotyrosine and 2100-
fold lower than the Km reported for iodotyrosine (50 µM,
bovine).4b Such alkylation was expected to enhance binding since
an iodo group would normally occupy this site. Ethyl and
isopropyl analogues of 2 were subsequently examined since these
larger alkyl substituents were previously shown to act as even
better mimics of the crucial 3′-iodo group in 3,3′,5-triiodothyro-
nine.20 For the deiodinase, the large alkyl groups were less
effective inhibitors and bound 3.6- and 17-fold weaker, respec-
tively, than the methyl derivative 2 (Table 1). Still, the N-
substituted amino acids exhibited substantial affinity as expected
for intermediate or transition-state analogues. In comparison,
substrate analogues such as 3-methyl- and 3-isopropyltyrosine are
very weak inhibitors of the enzyme with KI values greater than
880 µM.21
The pyridonyl analogues described here are particularly useful
for mimicking the nonaromatic tautomer of iodotyrosine, a critical
intermediate proposed for the enzyme-catalyzed deiodination of
this amino acid (Scheme 2).16 The π-electrons of this heterocyclic
system are substantially localized and overwhelmingly form the
pyridone rather than hydroxpyridine tautomer.22 The pyridonyl-
containing inhibitors also present structures that contrast to the
delocalized radical and anionic intermediates envisioned for
alternative23 homolytic (SRN1) and heterolytic (SNAR) mecha-
nisms. Additional analogues are currently under development for
further active-site characterization of iodotyrosine deiodinase and
related enzymes. The use of pyridonyl amino acids should also
be generally applicable to a variety of enzymes that catalyze other
aromatic substitution reactions including tyrosine-phenol lyase.24
phosphorus (red) in acetic anhydride. The resulting product was
neutralized with ammonia and crystallized to provide the desired
pyridonyl amino acid in an overall yield of 17%. Selective
methylation of this material with CH3I required initial formation
of its NR-BOC methyl ester derivative and then subsequent
deprotection to generate the product in a 57% yield from 1. Our
initial success with this compound as described below encouraged
us further to prepare the N-ethyl and N-isopropyl analogues (3
and 4, respectively) as well. The final protection and deprotection
steps were avoided when preparing these compounds (29% and
13% overall yields, respectively) by alkylating the intermediate
5-formyl-2-pyridone rather than the final pyridonyl amino acid
(Scheme 4).
Iodotyrosine deiodinase was solubilized from thyroid mi-
crosomes (porcine) using 1.5% Triton X-100 and assayed
according to the literature19 by 125I- release from [125I]-diiodoty-
rosine. This preparation of enzyme could be stored at 4 °C for a
month without significant loss of activity and exhibited a Km for
diiodotyrosine (9.3 µM, porcine) that was a little greater than
that (2.5 µM, bovine) measured after solubilizing the enzyme with
a lipase/protease combination.4b
The pyridonyl amino acids (1-4) were all observed to be
reversible and competitive inhibitors of diiodotyrosine turnover
under standard assay conditions. Time-dependent inactivation of
the enzyme was neither anticipated nor detected during incubation
with 2 (200 nM) for 120 min at 25 °C. Analogue 1 was an
(19) Rosenberg, I. N.; Goswami, A. Methods Enzymol. 1984, 107, 488-
500.
(20) Leeson, P. D.; Ellis, D.; Emmett, J. C.; Shah, V. P.; Showell, G. A.;
Underwood, A. H. J. Med. Chem. 1988, 31, 37-54.
(21) Green, W. Endocrinology 1968, 83, 336-347.
(22) (a) Beak, P. Acc. Chem. Res. 1977, 10, 186-192. (b) Gready, J. E.;
Hatton, P. M.; Sternhell, S. J. Heterocycl. Chem. 1992, 29, 935-946.
(23) (a) Zheng, Y.-J.; Ornstein, R. L. J. Am. Chem. Soc. 1997, 119, 648-
655. (b) Zheng, Y.-J.; Bruice, T. C. J. Am. Chem. Soc. 1997, 119, 3868-
3877.
(24) (a) Kiick, D. M.; Phillips, R. S. Biochemistry 1988, 27, 7333-7338.
(b) Sundararaju, B.; Antson, A. A.; Phillips, R. S.; Demidkina, T. V.; Barbolina,
M. V.; Gollnick, P. Dodson, G. G.; Wilson, K. S. Biochemistry 1997, 36,
6502-6510.
Acknowledgment. This research was supported in part by the National
Institutes of Health (DK-45783). We thank Mitsuhiro Koda for assistance
in preparing the transition-state (or intermediate) analogues and Tatiana
Boiko for preliminary characterization of the deiodinase.
Supporting Information Available: Synthesis and characterization
of the transition-state (or intermediate) analogues, 1-4, preparation and
assay of iodotyrosine deiodinase (porcine) (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA990693N