H. Tsutsumi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 741±743
743
ꢀ
Table 1. Kinetic parameters for the hydrolysis of p-nitrophenyl acetate catalyzed by EHb19 or Hb19 at 25 C in pH 6.0 phosphate buer solution
containing 1.25 vol% NMP
cat (10 3
�
s
� 1
)
K
m
(10 M)
� 4
k
cat/kun
kcat/K
(M
� 1 � 1
s
)
K
TS (10 M)
� 5
k
m
EHb19
Hb19
5.64
2.48
9.87
24.2
829
365
5.71
1.03
0.12
0.66
�
5
0
.66Â10
M for EHb19 and Hb19, respectively.
Komiyama, M.; Shigekawa, H. Comprehensive Supramolecular
Chemistry; Vol. 3, Cyclodextrins; Szejtli, J.; Osa, T., Eds.;
Pergamon: Oxford, New York, Tokyo, 1996, p. 401.
5. D'Souza, V. T.; Hanabusa, K.; O'Leary, T.; Gadwood, R.
C.; Bender, M. L. Biochem. Biophys. Res. Commun. 1985, 129,
EHb19 shows also about 5 times superior value to
Hb19. This result suggests that EHb19 could stabilize
the transition state of the substrate more eectively than
Hb19. Namely, the existence of the carboxylate of Glu6
enhances the catalytic activity by stabilizing the transi-
tion state of the substrate. The kcat/Km and KTS of the
simple imidazole attached b-CyD (6Im-b-CyD) were 1.7
7
6
27.
. Ekberg, B. F.; Anderson, L. I.; Mosbach, K. Carbohydr.
Res. 1989, 192, 111.
. For the review of de novo design of peptides, (a) DeGrado,
7
�
1
� 1
� 5
21
M
s
and 2.8Â10 M, respectively. Interestingly,
W. F.; Summa, C. M.; Pavone, V.; Nastri, F.; Lombardi, A.
Annu. Rev. Biochem. 1999, 68, 779. (b) Schneider, J. P.; Kelly,
J. W. Chem. Rev. 1995, 95, 2164.
the catalytic activity of EHb19 is similar to that of 6Im-
b-CyD, while both the CyD-peptide hybrids stabilize
transition state of the substrate more eectively than
8
2
9
. Chou, P. Y.; Fasman, G. D. Annu. Rev. Biochem. 1978, 47,
58.
. (a) Serrano, L.; Horovitz, A.; Avron, B.; Bycroft, M.;
6
Im-b-CyD. Especially, EHb19 stabilizes the transition
state of the substrate over 20 times than 6Im-b-CyD
did. Those results suggest that placing of the functional
groups at correct positions in the peptide backbone is
favorable to stabilize the transition state of the substrate.
Fersht, A. R. Biochemistry 1990, 29, 9343. (b) Dao-pin, S.;
Sauer, U.; Nicholson, H.; Matthews, B. W. Biochemistry 1991,
3
1
0, 7142.
0. Glutamate favors a-helical conformation than aspartate
(
almost identical.
11. Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno, A.; Toda,
F.; Suzuki, I.; Osa, T. J. Am. Chem. Soc. 1993, 115, 5035.
12. Fujii, N.; Otaka, A.; Ikemura, O.; Akaji, K.; Funakoshi,
S.; Hayashi, Y.; Kuroda, Y.; Yajima, H. J. Chem. Soc., Chem.
Commun. 1987, 274.
ref 8) and pK values of carboxyl group in Asp and Glu are
a
This is one of the successful examples of the arti®cial
enzyme having appropriately placed multiple functional
groups (an imidazole, a carboxylate, a binding site) on
an a-helix peptide. Also this new approach of ``cyclo-
dextrin±peptide hybrid'' may lead to novel possibility
for the design and construction of the better arti®cial
enzymes. The detailed study concerning the reaction
mechanism and substrate selectivity is now under way.
+
1
2
1
3. EHb19 m/z 2992.6 [(M+H) ], calc. 2992.1 and Hb19 m/z
934.3 [(M+H) ], calc. 2934.1
4. Due to poor solubility of the cyclodextrin±peptide hybrids
+
in water, tri¯uoroethanol (TFE) stock solutions of EHb19 or
�
�
3
Hb19 (2.0Â10 M) were prepared and diluted in phosphate
2
buer (5.0Â10 M). Final concentration of both catalysts is
Acknowledgements
� 5
2
1
.5Â10 M. pH was varied from 5.0 to 9.0.
5. Scholtz, J. M.; Qian, H.; York, E. J.; Stewart, J. M.;
This work was supported in part by The Japan Secu-
rities Scholarship Foundation and The Mochida Mem-
orial Foundation for Medical and Pharmaceutical
Research.
Baldwin, R. L. Biopolymers 1991, 31, 1463.
16. Tsutsumi, H.; Hamasaki, K.; Mihara, H.; Ueno, A.
unpublished data.
17. The UV absorption wavelengths of the p-nitrophenolate
were varied depending on the pH (320 or 400 nm).
1
8. For the study of ester hydrolysis, the stock solution of the
�
3
catalysts (2.0Â10 M) was prepared with N-methyl-2-pyrro-
References and Notes
lidone (NMP) and diluted in phosphate buer. Final con-
centration of both catalysts is 2.5Â10 M.
�
5
1
2
1
3
4
. Blow, D. M. Acc. Chem. Res. 1976, 9, 145.
. D'Souza, V. T.; Bender, M. L. Acc. Chem. Res. 1987, 20,
46.
. Bachovchin, W. W. Biochemistry 1986, 25, 7751.
. For the review of cyclodextrin as enzyme mimics: (a)
19. kcat is the ®rst order kinetic constant for the reaction from
the catalyst±substrate complex.
20. Kirby, A. J.; Kochi, J. K.; Kurtz, H. A.; Tee, O. S.;
Williams, R. V. Adv. Phy. Org. Chem. 1994, 29
21. (a) Lee, W-S.; Ueno, A. Chem. Lett. 2000, in press. (b)
Hamasaki, K.; Ueno, A. Chem. Lett. 1995, 859.
Breslow, R.; Dong, S. Chem. Rev. 1998, 98, 1997. (b)