E. Deguchi, K. Koumoto / Bioorg. Med. Chem. 19 (2011) 3128–3134
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2.2. Influence of chemical structure of zwitterionic solutes
As additives, not only zwitterionic solutes 1–9, but also glycerol,
sorbitol, and myo-inositol were used as references, because these
polyols have been reported to be useful additives for glucosidases.4
The initial velocity of the hydrolysis reaction was calculated from
the initial slope of the line generated by A405 versus incubation
time plots using the molar extinction co-efficient of p-nitrophenol
(10900 MÀ1 cmÀ1).
A comparison of the initial velocities of the hydrolysis reaction
by a-glucosidase (from all three sources) in the presence of zwit-
terionic solutes 1–9, glycerol, sorbitol, and myo-inositol (concen-
tration of all solutes was adjusted to 50 mM) is presented in
Figure 3, where the y-axis shows activity (activity = initial velocity
in the presence of solute/initial velocity in the absence of solute).
As shown in Figure 3, solute 6 possessed the highest activity for
Figure 1. Chemical structure of synthetic zwitterionic solutes.
a-glucosidases among all the solutes, and solute 5 showed the
second highest activity. Although the other solutes did not enhance
activity, the hydrolysis reaction was facilitated to some extent by
the addition of the synthetic zwitterionic solutes. In contrast, the
presence of polyols decreased activity.
To better understand the structural effects of the synthetic
solutes on the activity of a-glucosidase, we focused on a-glucosi-
dase isolated from B. stearothermophillus, as it appeared to be more
sensitive to the presence of the solutes. As shown in Figure 3, sol-
utes 4, 5, and 6 enhanced activity by 1.13 0.02, 1.54 0.07, and
2.74 0.02, respectively. These solutes have a common aliphatic
ammonium group and become bulkier from 4 to 5 to 6, indicating
that solutes possessing bulkier ammonium groups tend to acceler-
ate the enzymatic reaction more so than those possessing less
bulky groups. Subsequently, to compare functional groups intro-
duced outside the ammonium cation, we compared the activities
buffer. For example, Matholouthi et al. reported that carbohydrates
such as sucrose, trehalose, glucose, and sorbitol, which decreased
water activity in buffer solutions, intensified the enzymatic activity
of a protease (thermolysin) and two glycohydrolases (pullulanase
and inulinase).4 Rao and co-workers reported that the activity of
a glycohydrolase (xylanase) is enhanced by the addition of gly-
cine.6 However, enhanced activity was induced by only glycine
and not by other amino acids or derivatives. Considering these
reports, naturally-occurring metabolites, such as polyols and ami-
no acids, should have the potential to facilitate enzymatic reac-
tions. However, there have been few structural studies. Thus, we
investigated the effects of our metabolite analogs on the activity
of enzymes. We selected several hydrolases to study the effects
of our metabolite analogs since these hydrolases have already been
studied using naturally-occurring metabolites.4,6,9
of
a-glucosidase in the presence of solutes 4, 7, and 8, which all
possess a similar bulky ammonium cation, but differ in their other
functional groups. As a result, activity in the presence of solute 7
(1.05 0.04) was smaller than the others, indicating that the polar
oxygen atom located outside the ammonium cation reduced enzy-
matic activity. On the other hand, the activity was almost equal
when the ammonium cation possessed either a linear or cyclic ali-
phatic chain, as observed for solutes 4 and 8 (1.13 0.02 and
1.16 0.06, respectively). The present results indicate that in order
2. Results and discussion
2.1. Comparison of enzymatic hydrolysis in the absence and
presence of synthetic zwitterionic solutes
According to our previous study,14 solute 6 possessed the stron-
gest destabilization property for DNA duplexes. Therefore, we first
compared enzymatic reactions in the absence and presence of
to facilitate the hydrolysis reaction by a-glucosidase, bulky and ali-
phatic ammonium cations in zwitterionic metabolites are neces-
sary, and that there should be no polar moieties outside of the
aliphatic chain.
solute 6 (50 mM) using several hydrolases: two
different origin, from Bacillus stearothermophillus and Saccharomy-
ces cerevisiae, and a commercially available -glucosidase from
a-glucosidases of
a
yeast, a b-glucosidase from almonds; and an alkaline phosphatase
from calf intestines. Hydrolysis of the substrates (p-nitrophenyl-
2.3. Influence of solute concentration on the activity of
glucosidase
a-
a-D-glucopyranoside for a-glucosidase, p-nitrophenyl-b-D-gluco-
pyranoside for b-glucosidase, and p-nitrophenylphosphate for
alkaline phosphatase) in the absence and presence of solute 6 at
37 °C was followed by monitoring the absorbance at 405 nm
(A405) for p-nitrophenol produced during enzymatic hydrolysis
using a Multiskan microplate reader (Thermo Scientific Inc., Penn-
sylvania, USA).
Figure 2(a)–(e) shows plots of A405 versus incubation time for
various hydrolases. In all cases, the slopes in the presence of solute
6 were larger than those in the absence of solute 6, suggesting that
the zwitterionic solute facilitated the hydrolysis reaction, even
though the substrates and reaction mechanisms were different.
Using solutes 1, 4–6, and glycerol, sorbitol, and myo-inositol, we
next examined the dependence of enzymatic activity on the solute
concentration. As shown in Figure 4, solute 1, a naturally-occurring
metabolite, did not alter the activity even at concentrations up to
1000 mM. In contrast, solute 4, with an ethyl group instead of
the methyl group (solute 1) attached to the ammonium cation,
showed a maximum activity of 1.62 at above 800 mM. Moreover,
solute 5 showed a much higher maximum activity of 2.21 at
200 mM. The activity of
a-glucosidase was most significantly
affected in the presence of solute 6; the maximum activity reached
2.74 at 30–50 mM. These results revealed that as the length of the
alkyl chain increases, the effective concentration decreases and the
maximum activity increases. In contrast, all polyols
Furthermore, the degree of facilitation differed even for a-glucosi-
dases (Fig. 2(a)–(c)), implying that the acceleration effects of the
zwitterionic solutes may be related to the amino-acid sequence
or higher-order structure of the enzymes. To obtain further
insights into the acceleration mechanism by zwitterionic solutes,
decreased the activity of
a-glucosidase as the concentration
increased, indicating that the acceleration effect is characteristic
of these zwitterionic metabolite analogs. When the solute 6 con-
we investigated their effects using
a-glucosidases.
centration increased above 50 mM, the activity of a-glucosidase