Fig. 2 Hydrolysis of Boc-L-Phe-ONp and Boc-D-Phe-ONp (12.5 mM) in the presence of apomyoglobin (2–10 equiv.) in phosphate buffer (pH 7.0, 10 mM)
containing 1% dioxane at 4 °C: (a) plots of hydrolysis rate constant (kobs) vs. initial concentration of apomyoglobin, and (b) association constants of the
Michaelis complexes (Km21) and maximum rate constants (kcat).
2 (a) U. G. Wagner, N. Müller, W. Schmitzberger, H. Falk and C. Kratky,
J. Mol. Biol., 1995, 247, 326; (b) B. B. Lakshmi and C. R. Martin,
Nature, 1997, 388, 758.
3 For utilization of protein hydrophobic domains for catalysis, see: (a) M.
Nango, Y. Kimura, S. Kanda, Y. Ihara, J. Koga and N. Kuroki, Chem.
Lett., 1986, 229; (b) F. Hollfelder, A. J. Kirby and D. S. Tawfik, Nature,
1996, 383, 60; (c) H. Kuang and M. D. Distefano, J. Am. Chem. Soc.,
1998, 120, 1072.
4 Y. Ishida, K. Konishi, T. Aida and T. Nagamune, Chem. Eur. J., 1998,
4, 1148.
5 Y. Ishida, K. Konishi, T. Nagamune and T. Aida, J. Am. Chem. Soc.,
1999, 121, 7947.
6 Examples of enantioselective hydrolysis of amino acid esters, see:
(a) J.-J. Béchet, A. Dupaix and C. Roucous, Biochemistry, 1973, 12,
2566; (b) M. Tanihira and Y. Imanishi, Polym. J., 1983, 15, 499; (c) T.
Miyazawa, H. Iwanaga, S. Ueji, T. Yamada and S. Kuwata, J. Chem.
Soc., Chem. Commun., 1988, 1214; (d) R. Ueoka, Y. Matsumoto, K.
Harada, H. Akahoshi, Y. Ihara and Y. Kato, J. Am. Chem. Soc., 1992,
114, 8339; (e) ref 3a.
7 (a) Y. V. Griko, P. L. Privalov and S. Y. Venyaminov, J. Mol. Biol.,
1988, 202, 127; (b) F. M. Hughson, P. E. Wright and R. L. Baldwin,
Science, 1990, 249, 1544; (c) D. Barrick, F. M. Hughson and R. L.
Baldwin, J. Mol. Biol., 1994, 237, 588.
Boc- -Phe-ONp both showed a saturation signature (Fig. 2a),
indicating that the apoprotein and Boc-Phe-ONp form a
complex which serves as a reactive intermediate. Lineweaver–
D
Burk plots11,13 gave a maximum rate constant for the
L
-isomer
(kLcat) of 6.1 3 1021 min21, which is approximately 16 times
larger than that for the
-isomer (kD = 3.8 3 1022 min21
(Fig. 2b).14 On the other hand, the Km value for the hydrolysis
of Boc-
-Phe-ONp (KLm) was evaluated to be 1.5 3 1025 M,
D
)
cat
L
which is comparable to that of the
-isomer (KD = 1.4 3 1025
D
m
M).12 Thus, the high stereoselectivity of the hydrolysis is not
due to the enantioselective binding of the substrate (KL
/
21
m
21
KD
= 0.95) but is more likely due to the large difference in
m
reactivity between the
the heme pocket.
L
- and -isomers of Boc-Phe-ONp within
D
The enantioselectivity of the apomyoglobin-mediated hy-
drolysis was found to be highly sensitive to the substrate. For
example, when the N-protecting Boc group (R2) of the substrate
was replaced with benzyloxycarbonyl (Z-Phe-ONp, 12.5 mM),
the rate constant of the hydrolysis of the
L
-isomer with
apomyoglobin (62.5 mM) was considerably decreased (kL
obs
=
obs
= 3.1 3 1022 min21) to furnish a
5.5 3 1022 min21, kD
obs
8 H. Zemel, J. Am. Chem. Soc., 1987, 109, 1875.
kLobs+kD of only 1.8.15 Lower enantioselectivities were also
obs
9 F. W. J. Teale, Biochim. Biophys. Acta, 1959, 35, 543.
10 The reaction was followed by a change in absorbance at 400 nm due to
4-nitrophenolate anion (NpO2). The pseudo first-order rate constants
(kobs) were obtained according to the equation (1-[NpO2]/[Boc-Phe-
observed when Boc-PhGly-ONp (R1 = Ph, kLobs/kD = 1.8)16
obs
and Boc-Ala-ONp (R1 = Me, kLobs/kD
= 1.9)16 were the
substrates in place of Boc-Phe-ONp, although the
were again preferentially hydrolyzed.
L
-isomers
ONp]0) = e2kt
.
In conclusion, through the present studies on the utilization of
the apoprotein of horse-heart myoglobin as a potential chiral
catalyst, we have demonstrated a highly enantioselective
hydrolysis of a phenylalanine 4-nitrophenyl ester (Boc-Phe-
ONp), whose selectivity is one of the highest reported to date.
Exploration with other natural and mutated apoproteins is one
of the subjects worthy of further investigation for extending the
scope of reactions.
11 Electronic supplementary information (ESI) available: absorbance
b0/b008403o/
12 The second-order rate constant (k2) for the hydrolysis of Boc-Phe-ONp
with imidazole was 23 M21 min21, which is much smaller than the
catalytic constants (kcat/Km) for the hydrolysis of Boc-
L
-Phe-ONp (3.6
3 104 M21 min21) and Boc-
D
-Phe-ONp (0.24 3 104 M21 min21) with
apomyoglobin.
13 Examples of Michaelis–Menten kinetics using kobs instead of V0 in the
presence of an excess amount of mediators, see (a) V. Jubian, R. P.
Dixon and A. D. Hamilton, J. Am. Chem. Soc., 1992, 114, 1120; (b) R.
Hetting and H. J. Schneider, J. Am. Chem. Soc., 1997, 119, 5638.
We thank JSPS for financial support. Y. I. thanks the JSPS
Young Scientist Fellowship.
14 Maximum acceleration factors for the hydrolysis of the
L- and D-isomers
of Boc-Phe-ONp (kcat/kuncat) were 1200 and 73, respectively.
Notes and references
15 The uncatalyzed reaction was not examined due to a limited solubility
of Z-Phe-ONp.
1 (a) E. T. Kaiser and D. S. Lawrence, Science, 1984, 226, 505; (b) G. M.
Whitesides and C.-H. Wong, Angew. Chem., Int. Ed. Engl., 1985, 24,
617; (c) P. G. Schultz and R. A. Lerner, Acc. Chem. Res., 1993, 26, 391;
(d) R. O. Duthaler, Tetrahedron, 1994, 50, 1539.
16 Boc-PhGly-ONp: kL
= 1.3 3 1021 min21, kD
= 7.1 3 1022
obs
obs
min21, kuncat = 6.4 3 1024 min21; Boc-Ala-ONp: kL = 5.1 3 1022
obs
obs
= 2.7 3 1022 min21, kuncat = 3.1 3 1024 min21
.
min21, kD
134
Chem. Commun., 2001, 133–134