The new α-amino acid Nω-hydroxy-nor-L-arginine: A high-affinity inhibitor of arginase well adapted to bind to its manganese cluster

Full text of DOI:10.1021/ja970285o

4086
J. Am. Chem. Soc. 1997, 119, 4086-4087
The New r-Amino Acid
Nω-Hydroxy-nor-L-arginine: a High-Affinity
Inhibitor of Arginase Well Adapted To Bind to Its
Manganese Cluster
J. Custot,† C. Moali,† M. Brollo,† J. L. Boucher,†
M. Delaforge,† D. Mansuy,*,† J. P. Tenu,‡ and
J. L. Zimmermann§
Laboratoire de Chimie et Biochimie
Pharmacologiques et Toxicologiques, URA 400 CNRS
UniVersite´ Rene´ Descartes, 45 rue des Saints-Pe`res
75270 Paris Cedex 06, France
CNRS, URA 1116, Baˆt. 432
UniVersite´ Paris XI, 91405 Orsay, France
CEA/Saclay, De´partement de Biologie Cellulaire
et Mole´culaire, Section de Bioe´nerge´tique
Baˆt. 532, 91191 Gif Sur YVette, France
ReceiVed January 28, 1997
Mammalian arginases specifically require a Mn(II)-Mn(II)
cluster for their catalytic activity, the hydrolysis of L-arginine
(L-arg) to L-ornithine and urea.1 Quite recently, the crystal
structure of rat liver arginase has shown that the two Mn ions
are bridged by two carboxylate side chains of aspartates from
the protein and a water molecule (or hydroxide ion).2 It has
been proposed that L-arg binds in close proximity of the Mn-
cluster and that the transition state of the reaction is a tetrahedral
species resulting from nucleophilic attack of the metal-bridging
hydroxide at the guanidinium carbon of L-arg (Figure 1).2,3 Very
few data are presently available about the accessibility of the
Mn-cluster to molecules different from L-arg, and borate was
the only compound described so far to significantly modify the
EPR spectrum of arginase.4 Inhibitors often are interesting tools
to explore enzyme active sites. For arginase, for a long time
the best known inhibitor was L-valine, which exhibits a modest
Ki value (in the millimolar range).5 More recently, Nω-hydroxy-
L-arginine (NOHA)6 (Scheme 1), an intermediate in the bio-
synthesis of NO from L-arginine, and some Nω-hydroxy-L-R-
aminoacids7 have been shown to act as much more potent
inhibitors of arginases with Ki values in the 20-50 µM range.
A model has been proposed for the interaction of those inhibitors
with arginase, which postulated that they could be of the
transition state analog type, the N-OH group being able to
replace the hydroxo ligand of the arginase Mn-cluster.6a,7 On
the basis of this model, it appeared to us that Nω-hydroxy-nor-
L-arginine (nor-NOHA) should be a very good arginase inhibitor,
well suited to interact with the Mn cluster Via its N-OH
function (better than NOHA, Figure 1).
Figure 1. Schematic view of the postulated arginase transition state
and a proposed model for interaction of arginase with nor-HOHA and
NOHA.
Scheme 1. Synthesis of Nω-Hydroxy-nor-L-arginine
(nor-NOHA) and Formula of NOHA and homo-NOHAa
a Boc: -COOtBu; Z ) COOCH2Ph; (a) according to the general
procedure of ref 10; (b) conditions as in ref 11; (c) conditions of those
steps and the following ones as described for the synthesis of NOHA.8d
This paper reports preliminary results about (i) the first
synthesis of Nω-hydroxy-nor-L-arginine and its homolog Nω-
hydroxy-homo-L-arginine, (ii) a comparison of the inhibitory
effects of NOHA, homo-NOHA, and nor-NOHA toward argi-
nases, and (iii) EPR studies of the interaction of these
compounds with purified rat liver arginase. They show that
nor-NOHA not only is the best inhibitor (Ki ) 0.5 µM) but
also specifically modifies the arginase EPR spectrum.
Nω-hydroxy-homo-L-arginine (homo-NOHA) (Scheme 1)9
was synthesized from L-lysine following a procedure previously
described for the synthesis of NOHA.8 Nor-NOHA9 was
obtained from L-glutamine from reactions shown in Scheme 1
(15% overall yield). The key step was the decarboxylation of
NR-Boc-L-glutamine by sodium hypobromite to intermediate
NR-Boc-nor-L-ornithine which was readily protected as a Nδ-
† Universite´ Rene´ Descartes.
‡ Universite´ Paris XI.
§ CEA/Saclay.
(1) Reczkowski, R. S.; Ash, D. E. J. Am. Chem. Soc. 1992, 114, 10992-
10994.
(2) Kanyo, Z. F.; Scolnick, L. R.; Ash, D. E.; Christianson, D. W. Nature
1996, 383, 554-557.
(3) Reczkowski, R. S.; Ash, D. E. Arch. Biochem. Biophys. 1994, 312,
31-37.
(4) Khangulov, S. V.; Pessiki, P. J.; Barynin, V. V.; Ash, D. E.;
Dismukes, G. C. Biochemistry 1995, 34, 2015-2025.
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(6) (a) Daghigh, F.; Fukuto, J. M.; Ash, D. E. Biochem. Biophys. Res.
Commun. 1994, 202, 174-180. (b) Boucher, J. L.; Custot, J.; Vadon, S.;
Delaforge, M.; Lepoivre, M.; Tenu, J. P.; Yapo, A.; Mansuy, D. Biochem.
Biophys. Res. Commun. 1994, 203, 1614-1621.
(9) Both homo- and nor-NOHA were characterized by 1H and 13C NMR
spectroscopies: δ13C(D2O) 24.3, 30.3, 32, 43.5, 55.9, 161.6, 175.3 and 31.8,
40.1, 53.2, 161.5, 174.2, respectively. Mass spectrometry m/z (ES): 205.23
and 177.18 (M + H+), respectively.
(7) Custot, J.; Boucher, J. L.; Vadon, S.; Guedes, C.; Dijols, S.; Delaforge,
M.; Mansuy, D. J. Biol. Inorg. Chem. 1996, 1, 73-82.
(8) (a) Feldman, P. L. Tetrahedron Lett. 1991, 32, 875-878. (b) Wallace,
G. C.; Fukuto, J. M. J. Med. Chem. 1991, 34, 1746-1748. (c) Bailey, D.
M.; DeGrazia, C. G.; Lape, H. E.; Frering, R.; Fort, D.; Skulan, T. J. Med.
Chem. 1973, 16, 151-156. (d) Vadon, S.; Custot, J.; Boucher, J. L.; Mansuy,
D. J. Chem. Soc., Perkin Trans. 1 1996, 645-648.
(10) Rudinger, J.; Poduska, K.; Zaoral, M. Coll. CzechosloV. Chem.
Commun. 1960, 25, 2022-2028.
(11) Chevallet, P.; Garrouste, P.; Malawska, B.; Martinez, J. Tetrahedron
Lett. 1993, 34, 7409-7412.
S0002-7863(97)00285-0 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 17, 1997 4087
enhanced. Other changes concern troughs at high-field related
to the zero-field splitting structure and observed between 460
and 700 mT. Three troughs at 505, 546, and 606 mT found
for native arginase were replaced by a new trough centered at
542 mT. Clear changes were already obtained after addition
of 1 equiv of nor-NOHA per Mn-dimer (1 mM); further
additions (up to 2 equiv) only slightly modified the spectrum
except for an increase of a six-line signal centered at 335 mT
with a 90 G hyperfine coupling constant (Figure 2B). The latter
signal could be due to loosely bound Mn ions (or Mn ions not
coupled as in native arginase)13 as it disappeared after ultrafil-
tration of the incubate. Moreover, the EPR spectrum of the
recovered sample was almost identical to the one obtained after
addition of 2 equiv of nor-NOHA to arginase, except for the
region around 335 mT which now showed a signal with at least
11 lines and hyperfine constants of 45 G that was masked by
the six-line (90 G) signal due to loosely bound Mn (Figure 2C).
These data suggest that nor-NOHA stoichiometrically interacts
with the Mn-cluster of arginase and leads to a marked change
of its EPR spectrum which is indicative of a change in the
Mn‚‚‚Mn distance4 and could be due to the replacement of the
arginase µ-OH ligand by its N-OH moiety. Upon addition of
excess nor-NOHA, nonspecific demetallation of the active site
could occur. Similar experiments performed with homo-NOHA
failed to induce any EPR spectral change on arginase, whereas
preliminary studies performed with NOHA only led to a marked
increase of the signal centered at 335 mT with a 90 G hyperfine
coupling constant. However, the other spectral changes ob-
served after addition of 1-2 equiv of nor-NOHA to arginase
(Figure 2) were not observed with either NOHA or homo-
NOHA.
The aforementioned results show that the length of the chain
linking the amino acid and N-hydroxyguanidine functions of
NOHA is very important for recognition by arginase. Addition
of a CH2 moiety (homo-NOHA) led to a dramatic decrease of
this recognition (75-fold increase of IC50), whereas removal of
one CH2 group markedly improved it (20-fold decrease of Ki).
This particularly high affinity of nor-NOHA for arginase appears
to be correlated to its specific interaction with the Mn-cluster
of the enzyme active site, which occurs with a 1:1 stoichiometry,
as suggested by EPR data. Additional experiments are required
in order to determine the molecular nature of the interaction of
nor-NOHA with arginase. A possible explanation for the
specific effects of this molecule is that it acts, as previously
assumed (Figure 1), by replacement of the H2O (or OH) bridging
ligand of the Mn-cluster by its N-hydroxy function. Molecular
models suggest that it would be better than NOHA to play this
role (Figure 1). The new R-amino acid, nor-NOHA, is the most
potent inhibitor of arginase (both from rat liver or mouse
macrophages) reported so far. Its use for better understanding
the physiological roles of arginases in Vitro and in ViVo is
underway.
Figure 2. Effects of nor-NOHA on the EPR spectrum of rat liver
arginase: (A) 1 mM arginase in 50 mM Tris-HCl buffer, pH 7.4, at 15
K (microwave frequency 9.42 GHz); (B) spectrum after addition of 2
mM nor-NOHA; (C) spectrum of sample B after ultrafiltration through
a Bio-spin column.
benzyloxycarbonyl (Boc) derivative before esterification to the
corresponding tert-butyl ester.
nor-NOHA was found to be much more potent than NOHA
and homo-NOHA to inhibit the rat liver arginase-dependent
hydrolysis of L-arg (respective IC50 of 2 ( 0.5, 40 ( 10, and
3000 ( 500 µM at pH 7.4).12 nor-NOHA is also a much better
inhibitor of the arginase activity of mouse macrophages than
NOHA (IC50 of 50 ( 10 and 450 ( 50 µM, respectively).
Lineweaver-Burk plots of data from incubations of purified
rat liver arginase with L-arg and either nor-NOHA or NOHA,
at pH 7.4, clearly indicated that the two latter compounds were
competitive inhibitors with respect to L-arg (Ki ) 0.5 ( 0.1
and 10 ( 2 µM, respectively). Experiments based on prein-
cubations of arginase with nor-NOHA showed that this com-
pound is, as NOHA,6 a reversible inhibitor. Finally, in double-
inhibition experiments performed with arginase, NOHA, and
nor-NOHA, plots of 1/activity as a function of nor-NOHA and
NOHA concentrations led to parallel curves, with a ratio of
about 20 between the two Ki values (data not shown). All these
results indicate that both compounds are reversible, competitive
inhibitors of arginase, with nor-NOHA having a much better
affinity.
Stepwise additions of nor-NOHA to purified rat liver arginase
led to a progressive change of the EPR spectrum of this
metalloprotein (Figure 2). Almost all EPR features previously
reported for native arginase4 were changed; for instance, the
signal set at 272 mT markedly decreased and that at 183 mT
disappeared, while the intensity of the 413 mT signal was
JA970285O
(12) Rat liver arginase was purified as described in ref 1 and its activity
measured as reported in ref 7.
(13) Ash, D. E.; Schramm, V. L. J. Biol. Chem. 1982, 257, 9261-9264.