Metal ion induced allosteric transition in the catalytic activity of an artificial phosphodiesterase

Article abstract of DOI:10.1039/b314032f

An artificial phosphodiesterase (1) bearing two kinds of metal binding sites, a catalytic site and a regulatory bipyridine site showed a unique allosteric transition in the catalytic activity against the metal concentration.

Full text of DOI:10.1039/b314032f

Metal ion induced allosteric transition in the catalytic activity of an
artificial phosphodiesterase†
Shinji Takebayashi, Masato Ikeda, Masayuki Takeuchi* and Seiji Shinkai*
Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka
812-8581, Japan. E-mail: taketcm@mbox.nc.kyushu-u.ac.jp. E-mail: seijitcm@mbox.nc.kyushu-u.ac.jp;
Fax: +81-92-642-3611
Received (in Cambridge, UK) 5th November 2003, Accepted 5th December 2003
First published as an Advance Article on the web 21st January 2004
An artificial phosphodiesterase (1) bearing two kinds of metal
binding sites, a catalytic site and a regulatory bipyridine site
showed a unique allosteric transition in the catalytic activity
against the metal concentration.
(more than 2 equiv. Zn2+). The absorbance of DPA moieties
changed almost linearly upon addition of 0 to 1 equiv. Zn2+ and 1
to 2 equiv. Zn2+. This result clearly shows that the first two Zn2+
ions are bound to the DPA moieties quantitatively to produce
1·(Zn2+)2 under these conditions. At [Zn2+] higher than 2 equiv., a
typical shift of the absorption band of the Bpy moiety from 284.6
nm to 312.0 nm was observed, indicating that Bpy·Zn2+ is formed
at this stage. A plot of the absorbance at 312.0 nm against Zn2+
concentration showed a saturation behaviour and the association
constant for the formation of 1·(Zn2+)3 from 1·(Zn2+)2 was
evaluated to be 9.1 x 102 M21. Similar results were obtained for the
1 (1.0 mM) and Cu2+ system, where 2 equiv. of Cu2+ are bound to
the DPA moieties in 1 quantitatively. The association constants for
the formation of 1·(Cu2+)3 and 12·(Cu2+)5 from 1·(Cu2+)2 were
evaluated to be 6.3 3 104 M21 and 1.0 3 108 M22, respectively.
The design of artificial allosteric systems is of great significance for
regulating the catalytic activities and complexation properties of
artificial receptors.1 We have been interested in the exploitation of
homotropic allosteric systems, which show the nonlinear amplifica-
tion of binding events and chemical signals.2 This phenomenon is
useful to control the activities of an artificial receptor in an OFF–
ON switching manner at a threshold condition.1,2 Allosteric
molecular recognition systems have been studied well, whereas
studies on artificial allosteric catalytic systems are still very rare.3,4
Furthermore, to our best knowledge, there is no report that the same
metal ion plays both roles of a catalytic metal ion and an effector
metal ion, giving rise to an allosteric transition phenomenon.4,5
Such a novel system would show a unique catalytic activity
response depending on the metal ion concentration.
There is considerable interest in creating artificial phosphodies-
terases because of their potential application in gene therapy. For
this purpose, numerous catalysts containing essential metal ions
have been explored.6 Here we designed compound 1 as an allosteric
artificial phosphodiesterase, which has two different types of metal
ion binding sites, viz. a 2,2A-bipyridine (Bpy) as a regulatory site
and 2,2A-dipicolylamine (DPA) as catalytic sites.7 The DPA
moieties have higher Zn2+ or Cu2+ affinities than the Bpy moiety.8
Hence one can predict that the first two metal ions are bound to two
DPA moieties in 1 to produce 1·(M2+)2 (M2+: Zn2+ or Cu2+). The
regulatory site, the Bpy moiety, adopts mostly a transoid conforma-
tion because of the repulsion between lone-pairs of nitrogen
atoms,9 where the amine distance between DPA is estimated to be
0.84 nm (catalytically less active). The metal ion coordination to
Bpy can induce a conformational change from transoid to cisoid
enforcing an alignment of the DPA sites at a distance of 0.48 nm;
that is ‘an allosteric transition’ (Fig. 1). Reference compounds 2
and 3 should not show such an allosteric transition phenomenon.
The complexation behaviour of 1 (0.4 mM) with Zn2+ (as a
perchlorate salt) in ethanol/water (HEPES, 16 mM) = 1:2 v/v
solution at pH 7.7 and 25 °C was monitored by photometric
titration. Binding processes of Zn2+ to 1 were accompanied by three
steps of spectral changes with isosbestic points observed at 293.2
nm (0 to 1 equiv. Zn2+), 296.8 nm (1 to 2 equiv. Zn2+) and 297.4 nm
1
These complexation process were confirmed by ESI-MS and H-
NMR spectroscopies (see supplementary information†).
We employed 2-hydroxypropyl-p-nitrophenyl phosphate
(HPNP) as a substrate.10 Kinetic experiments were conducted on
the basis of the release rate of p-nitrophenol from HPNP by
monitoring the increase in the absorbance at 406 nm. At first, the
kinetic studies at varying Zn2+ and Cu2+ concentrations were
performed and pseudo-first-order rate constants (kobs) were eval-
uated for 1 and 2 (Figs. 2 and 3). Very interestingly, in the case of
1, there was a further significant increase in the catalytic activity
upon addition of more than 2 equiv. metal ion, whereas such
enhancement was not observed for 2 without the regulatory site
Fig. 1 Schematic representation of allosteric transition of 1.
† Electronic supplementary information (ESI) available: synthesis of 1,
characterization of complexes by 1H-NMR and ESI-MS spectroscopies and
the analysis of the kinetic data. See http://www.rsc.org/suppdata/cc/b3/
b314032f/
Fig. 2 Plots of pseudo-first-order rate constants (kobs) for the hydrolysis of
HPNP (0.8 mM) at various Zn2+ concentrations in 33% ethanol/water
(HEPES, 25 mM): (a) [1] = 0.4 mM, (b) [2] = 0.4 mM, pH 7.7 at 25
°C.
420
C h e m . C o m m u n . , 2 0 0 4 , 4 2 0 – 4 2 1
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

(Figs. 2 and 3). In Fig. 2, the rate constant for 1 with 8 equiv. Zn2+
is 3.3 times larger than that for 1 with 2 equiv. Zn2+ (1·(Zn2+)2). The
saturation behaviour observed for 1 in Fig. 2 is complementary to
the results of photometric titration. In the case of 1 and Cu2+, 19.4
times larger kobs value was evaluated for 1 with 3 equiv. Cu2+
against 1 with 2 equiv. Cu2+ (1·(Cu2+)2) (Fig. 3). For 1 with 3 equiv.
Cu2+, the ratio of [1·(Cu2+)3]/[12·(Cu2+)5]/[1·(Cu2+)2] is estimated
to be 81/11/8 by the association constants between Cu2+ and the
Bpy moiety in 1.11 These results indicate that 1·(M2+)3 and/or
12·(M2+)5 have higher catalytic activity than 1·(M2+)2.
In order to evaluate the contribution of the Bpy·Zn2+ complex to
the hydrolysis of HPNP, kobs values with control compounds 3 and
4 were measured. It was found that kobs value of 6.6 3 1025
s21obtained from a mixture of 2·(Zn2+)2 and 4·Zn2+ is almost same
as that obtained form 2·(Zn2+)2. In addition, the kobs value of 3.5 3
1025 s21 for 3·(Zn2+)2 is 5.8 times smaller than that for 1·(Zn2+)3.
These results show that inter- or intra-molecular reaction catalysed
by Bpy·Zn2+ and Dpa·Zn2+ complexes is not so effective for the
hydrolysis of HPNP. Similar results were obtained from Cu2+ (see
supplementary information†). The increase in the catalytic activity
is ascribed, therefore, to the allosteric conformational transition of
1 induced by the coordination of effector metal ion to the Bpy
moiety as shown in Fig. 1.
Michaelis–Menten kinetic parameters were evaluated from
saturation kinetic experiments to obtain a further insight into the
rate enhancement observed with 1. Saturation kinetic curves were
obtained from Zn2+ and Cu2+ catalysts: (a); [1] = 0.4 mM, [Zn2+]
= 0.8 mM, (b); [1] = 0.4 mM, [Zn2+] = 2.0 mM, (c); [1] = 1.0
mM, [Cu2+] = 2.0 mM, (d); [1] = 1.0 mM, [Cu2+] = 3.0 mM. A
Lineweaver–Burk plot was, then, applied to calculate the Michae-
lis–Menten constant (Km) and the catalytic constant (kcat) (see
supplementary information†). The results are summarized in Table
1. A significant increase in the value of kcat (4.1 times for Zn2+ and
55 times for Cu2+) was obtained from the conditions (b) and (d)
compared with (a) and (c). These results indicate that the
conformational change of 1 induced by the third M2+ complexation,
that is, allosteric transition, enhances the rate of hydrolysis but not
by a change in the substrate affinity. This should be a consequence
of the preferable preorganisation of two DPA·M2+ complex units
toward the hydrolysis, which would facilitate the attack of
intramolecular hydroxyl ion sitting on M2+ to the substrate.5,6
In conclusion, we have demonstrated that compound 1 bearing
two types of Zn2+ or Cu2+ binding sites shows a novel allosteric
response in the catalytic activities toward the hydrolysis of
phosphodiester. We anticipate, therefore, that such a supramo-
lecular catalytic system would further produce intelligent artificial
systems responding to various kinds of chemical stimuli.
This work is partially supported by a Grant-in-Aid for the 21st
Century COE Program, “Functional Innovation of Molecular
Informatics” from the Ministry of Education, Culture, Science,
Sports and Technology of Japan. We thank Prof. Itaru Hamachi, Dr.
Akio Ojida and Mr. Masa-aki Inoue at Kyushu University for
helpful discussions. M.T. and S.T. thank Prof. Fumito Tani and Mr.
Jun Hagiwara for ESI-MS measurements. M.I. thanks JSPS for the
Research Fellowship for Young Scientists for financial support.
Notes and references
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2 (a) S. Shinkai, M. Ikeda, A. Sugasaki and M. Takeuchi, Acc. Chem.
Res., 2001, 34, 494; (b) M. Takeuchi, M. Ikeda, A. Sugasaki and S.
Shinkai, Acc. Chem. Res., 2001, 34, 865; (c) M. Ikeda, M. Takeuchi and
S. Shinkai, J. Synth. Org. Chem. Jpn., 2002, 60, 1201.
3 T. Tozawa, S. Tokita and Y. Kubo, Tetrahedron Lett., 2002, 43,
3455.
4 Krämer et al. reported artificial allosteric catalysts, where ligands bound
regulatory metal ions in advance: I. O. Fritsky, R. Ott, H. Pritzkow and
R. Krämer, Chem. Eur. J., 2001, 7, 122.
5 Significant rate enhancement was observed for trinuclear Zn2+ complex
(as a catalytic metal ion) for the hydrolysis of diribonucleotides: see ref.
6b.
6 For recent reviews (a) M. Molenveld, J. F. J. Engbersen and D. N.
Reinhoudt, Chem. Soc. Rev., 2000, 29, 75; (b) M. Yashiro, A. Ishikubo
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Berni, C. Caltagirone, F. A. Devillanova, F. Isaia, A. Garau, C. Giorgi,
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7 A. Ojida, M.-a. Inoue, Y. Mito-oka and I. Hamachi, J. Am. Chem. Soc.,
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8 (a) J. K. Romary, J. D. Barger and J. E. Bunds, Inorg. Chem., 1968, 7,
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9 (a) A. Goller and U. W. Grummt, Chem. Phys. Lett., 2000, 321,
401.Rebek and co-workers have reported a significant reaction rate
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tion with 2,2A-bipyridine (b) J. Rebek, Jr., T. Costello and R. Wattley, J.
Am. Chem. Soc., 1985, 107, 7487 see also ref. 1(b); Recently, Lehn and
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10 D. M. Brown and D. A. Usher, J. Chem. Soc., 1965, 6588.
11 For 1 with 3.5 equiv. Cu2+, the ratio of [1·(Cu2+)3]/[12·(Cu2+)5]/
[1·(Cu2+)2] is estimated to be 92/5/3 by the association constants
between Cu2+ and the Bpy moiety in 1. The decrease in the kobs value
upon addition of more than 3 equiv. Cu2+ is ascribed to the formation of
precipitate under these conditions.
Fig. 3 Plots of pseudo-first-order rate constants (kobs) for the hydrolysis of
HPNP (1.0 mM) at various Cu2+ concentrations in 33% ethanol/water
(HEPES, 25 mM): (a) [1] = 1.0 mM, (b) [2] = 1.0 mM, pH 7.7 at 25 °C.
Inset is an enlarged view for 2 and Cu2+
.
Table 1 Michaelis–Menten kinetic parameters
102·kcat/s21
102·Km/M
(a) 1 with 2 equiv. Zn2+
(b) 1 with 5 equiv. Zn2+
(c) 1 with 2 equiv. Cu2+
(d) 1 with 3 equiv. Cu2+
0.051
0.19
0.31
0.36
1.27
0.21
0.026
1.43
C h e m . C o m m u n . , 2 0 0 4 , 4 2 0 – 4 2 1
421