Optimum conformational flexibility of subtilisin to maximize the enantioselectivity for subtilisin-catalysed transesterification in an organic solvent with an addition of dimethyl sulfoxide

Article abstract of DOI:10.1039/b101269j

For subtilisin-catalysed transesterification of the racemic esters in i-octane containing dimethyl sulfoxide as an additive, the relationship between the enantioselectivity and the conformational flexibility of subtilisin estimated from the ESR spectrosco

Full text of DOI:10.1039/b101269j

Optimum conformational flexibility of subtilisin to maximize the
enantioselectivity for subtilisin-catalysed transesterification in an
organic solvent with an addition of dimethyl sulfoxide
Keiichi Watanabe,a Takashi Yoshidab and Shin-ichi Ueji*ab
a The Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan
b Division of Natural Environment and Bioorganic Chemistry, Faculty of Human Development and
Sciences, Kobe University, Nada, Kobe 657-8501, Japan. E-mail: ueji@kobe-u.ac.jp;
Fax: +81-78-803-7761
Received (in Cambridge, UK) 8th February 2001, Accepted 21st May 2001
First published as an Advance Article on the web 20th June 2001
For subtilisin-catalysed transesterification of the racemic
esters in i-octane containing dimethyl sulfoxide as an
additive, the relationship between the enantioselectivity and
the conformational flexibility of subtilisin estimated from
the ESR spectroscopic study provides the first experimental
evidence that the enzyme has the optimum flexibility to
produce the maximal enantioselectivity toward the given
substrates.
The effects of the additives (0.45 vol%) on the initial rates as
a measure of the enzymatic activity for subtilisin-catalysed
transesterification of 1 in i-octane were investigated. When a
small amount of DMSO (0.45 vol%) was added to the reaction
medium, the initial rate for the correctly binding S enantiomer
was found to be dramatically enhanced (Vs
20 nmol h21 mg21), as compared with those for the addition of
other general additives such as water (Vs
=
=
0.21 nmol h21 mol21) or polar organic solvents (DMF, THF,
acetone and acetonitrile), although the reaction for no additive
conditions did not proceed at all. The effect of DMSO on the
enantioselectivity enhancement was also observed for sub-
tilisin-catalysed hydrolysis of 1 in aqueous buffer containing
DMSO.5
The ability of enzymes to discriminate between enantiomers has
been applied to the optical resolution of racemates in organic
syntheses.1 Among the strategies to improve the enzyme’s
enantioselectivity, organic chemists and enzymologists often
employ various additives for enzyme-catalysed reactions in
organic solvents,2 because of their simplicity of use. Although
the variation of the enzyme’s flexibility caused by an additive,
such as a small amount of water in organic solvents, is
anticipated to control its enantioselectivity,3 definite informa-
tion is lacking as to the relationship between the enzyme’s
flexibility and its enantioselectivity for enzyme-catalysed
reactions. According to research done so far, the only reported
observation is that the effect on enantioselectivity by the
variation of organic solvents is related to the enzyme’s
flexibility, estimated from time-resolved fluorescence aniso-
tropic study.4
In this communication, for subtilisin-catalysed transester-
ification in i-octane, we wish to report the mechanism of
enantioselectivity enhancement caused by addition of dimethyl
sulfoxide on the basis of the relationship between the initial
rates for each enantiomer of the substrates used here and the
conformational flexibility of subtilisin estimated from ESR
spectroscopic study. Furthermore, the relationship established
reveals that the optimum conformational flexibility of subtilisin
gives the maximal enantioselectivity.
For subtilisin-catalysed transesterification of ethyl (R)- or
(S)-2-(4-ethylphenoxy)propionate 1 with n-butyl alcohol in i-
octane, we investigated the behaviour of the initial rates for each
enantiomer of 1 caused by addition of a small amount of various
additives such as water or polar organic solvents (Scheme 1),
because this method of enantioselectivity improvement by
additives is the simplest one. In a typical subtilisin-catalysed
transesterification, the substrate 1 (0.025 mmol) and n-butyl
alcohol (0.15 mmol) were added to dry i-octane (2 ml)
containing the additives (0–0.60 vol%), followed by ultrasonic
dispersion, and then addition of subtilisin (10 mg).† The
reaction mixture was shaken (170 strokes min21) at 37 °C.
In order to elucidate the optimum amount of added DMSO,
the initial rates for each enantiomer of 1 for subtilisin-catalysed
transesterification in i-octane were determined for a range of
added DMSO (0–0.60 vol%). As is seen in Table 1, the initial
rate for the correctly binding S enantiomer was dramatically
enhanced by DMSO added to the reaction medium, as compared
with that for the incorrectly binding R enantiomer. In particular,
upon the addition of 0.45 vol% of DMSO, subtilisin displayed
the largest initial rate for the S enantiomer, thus resulting in the
maximal enantioselectivity (VS/VR = 9.6). A serious decrease
in the enzymatic activity, however, was produced by addition of
an excess amount of DMSO (0.60 vol%). This is probably
because the increased flexibility of subtilisin caused by the
excess addition of DMSO does not contribute to accommodat-
ing the substrate 1 into the subtilisin’s binding site, thus leading
to the decrease of the enzymatic activity accompanying the loss
of the enantioselectivity. This assumption is also supported by
the discussion below on the basis of the results obtained from
the ESR spectroscopic study.
Furthermore, in order to investigate the DMSO effect on the
other substrate, methyl mandelate 2 was submitted to the model
reaction. For subtilisin-catalysed transesterification in i-octane,
subtilisin preferentially catalysed the R enantiomer of 2. The
maximal enantioselectivity (VR/VS = 7.0) with an increase of
Table 1 Effects of DMSO on the enantioselectivity (VS/VR) and the initial
rates for each enantiomer of ethyl 2-(4-ethylphenoxy)propionate 1 for
subtilisin-catalysed transesterification in i-octane
Initial rate (1021 nmol h21 mg21
)
Amount (vol%)
VS
VR
VS/VR
0
0
0
17
105
201
65
0
0
9.9
26
21
19
10
—
—
0.150
0.300
0.375
0.450
0.500
0.600
1.7
4.0
9.6
3.4
1.4
Scheme 1 Subtilisin-catalysed transesterification of ethyl 2-(4-ethylphen-
oxy)propionate 1 with n-butyl alcohol in i-octane.
14
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Chem. Commun., 2001, 1260–1261
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b101269j

Fig. 1 Typical ESR spectrum of the spin-labeled subtilisin in i-octane; (A)
no additive condition, (B) 0.45 vol% DMSO.
Fig. 3 The relationship between the Hi/(Ha + Hi) value and the initial rates
for subtilisin-catalysed transesterifications of 1 and 2 in i-octane, 5: 1-(R),
2: 1-(S), -: 2-(R), 8: 2-(S).
the enzymatic activity was produced by addition of 0.375 vol%
of DMSO. Therefore, our approach using DMSO as the additive
was found to be valid for the improvement of the enantiose-
lectivity for subtilisin-catalysed reaction in an organic solvent,
although there is the difference in the optimum amount of
DMSO between 1 and 2 to obtain the maximal enantiose-
lectivity.
are depicted in Fig. 3. The initial rate for the correctly binding
S enantiomer was significantly enhanced by an increase of the
Hi/(Ha + Hi) value. On the other hand, for the incorrectly
binding R enantiomer, the initial rate is almost unchanged, in
spite of the increase of the Hi/(Ha + Hi) value. For subtilisin-
catalysed transesterification, the variation of the subtilisin’s
conformational flexibility caused by addition of DMSO is found
to be ascribed to the acceleration of the initial rate for the
correctly binding S enantiomer, as compared with that for the
incorrectly binding R enantiomer. Thus, the larger value of the
ratio of the initial rates, arising from the marked difference in
the flexibility effect on the initial rates for each enantiomer, is
significantly responsible for the enhancement of the subtilisin’s
enantioselectivity.
A serious drop in the initial rates, however, was produced by
a small increase of the Hi/(Ha + Hi) value from the optimum
flexibility to produce the largest initial rate (Fig. 3). This result
is explained by assuming that the subtilisin’s flexibility caused
by the excess addition of DMSO does not induce the stable
association between the substrate 1 and the subtilisin’s binding
site. Thus, subtilisin is found to display the optimum flexibility
to produce the maximal enantioselectivity and the enzymatic
activity toward the substrates used here. Furthermore, as is seen
in Fig. 3, the change of the substrate from 1 to 2 shows the
difference in the optimum flexibility to maximize the enantiose-
lectivity [(Hi/(Ha + Hi) for the largest VS/VR value of 1] > [Hi/
(Ha + Hi) for the largest VR/VS of 2], which suggests that the
optimum conformational flexibility is responsible for the
substrate’s structure. Our first observation offers an important
insight into the mechanism of the enantioselectivity enhance-
ment for the enzyme-catalysed reactions under the various
reaction conditions.
The remarkable enhancement of the enantioselectivity ob-
served is anticipated to be strongly affected by the change of the
subtilisin’s conformational flexibility caused by addition of
DMSO. This view promoted us to investigate the relationship
between the initial rates of each enantiomer of 1 and 2, and the
subtilisin’s conformational flexibility estimated from ESR
spectroscopy. The ESR measurement was carried out under the
same conditions as that for the subtilisin-catalyzed transester-
ification of 1 and 2, using a spin-labeled subtilisin with 1-oxy-
2,2,6,6,-tetramethyl-4-piperidinyl ethoxyphosphorofluoridate
prepared by the known method.6 The spin-labeled subtilisin was
indicated by MALDI-TOF MS, in which a fragment (27543)
was found which almost corresponded to the sequence of
subtilisin (27287) plus the weight of a spin-label (282) less the
weight of F (19) and H (1). The spin-labeled subtilisin showed
a decrease of enzymatic activity for our model reaction, due to
the inhibition by the spin label attached to the active site serine.
Fig. 1 shows a typical ESR spectrum, in which two parts of the
spectrum are arbitrarily labeled Ha and Hi, respectively. The
degree of the subtilisin’s conformational flexibility can be
monitored roughly by the change in the ratio of the peak height
of Hi to (Ha + Hi),7 because each peak of Ha and Hi represents
the anisotropy and the isotropy of the subtilisin’s spin-label,
respectively.8 Thus, the increase of the Hi/(Ha + Hi) value
reflects that the subtilisin’s conformation becomes more
flexible. Fig. 2 shows the variation of the Hi/(Ha + Hi) value
estimated from the ESR spectra in i-octane as a function of the
amount of DMSO, in which the increased amount of DMSO in
i-octane is found to increase the conformational flexibility of
subtilisin. In addition, under other additive conditions that give
poor enantioselectivity and low enzymatic activity, the ESR
signal showed the characteristics of a conformationally rigid
enzyme (Hi/(Ha + Hi) = 0.10–0.27).
Notes and references
† Because the presence of water is important in the activity of enzymes in
non-polar solvents, i-octane, n-butyl alcohol, and additives used here were
dried over Molecular Sieves 4 Å. For our model reactions, however, the
enzymatic activity was insensitive to the addition of water (0–0.9 vol%) into
i-octane.
The plots of the initial rates for each enantiomer of 1 for
subtilisin-catalysed transesterification in i-octane containing a
small amount of DMSO as a function of the Hi/(Ha + Hi) value
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Sugahara, S. Sugiyama and H. Hayashi, Biotechnol. Lett., 1995, 17, 1161;
H. Kitaguchi, I. Itoh and M. Ono, Chem. Lett., 1990, 1203.
4 J. Broos, A. J. W. G. Visser, J. F. J. Engbersen, W. Verboom, A. Hoek and
D. N. Reinhoudt, J. Am. Chem. Soc., 1995, 117, 12657.
5 K. Watanabe and S. Ueji, Biotechnol. Lett., 2000, 22, 599.
6 J. D. Morrisett and C. A. Broomfield, J. Biol. Chem., 1972, 247, 7224.
7 L. J. Berliner, Biochemistry, 1972, 11, 2921.
8 P. P. Wangikar, P. C. Michels, D. S. Clark and J. S. Dordick, J. Am.
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Fig. 2 The variation of the Hi/(Ha + Hi) value estimated from the ESR
spectra in i-octane as a function of the amount of DMSO.
Chem. Commun., 2001, 1260–1261
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