# The Crystal Water Affect in the Interaction between the Tenebrio Molitor Alpha-Amylase and Its Inhibitor

Authors: Zhu Zhi-Fei; Ning Ting-Ting; Xu Zu-Min; Zhang Ge-Xin; Ma Yan-He
Journal: Bioinorganic Chemistry and Applications
 (2008)

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## Abstract

Molecular dynamics simulation of the interaction between the Tenebrio molitor alpha-amylase and its inhibitor at different proportion of crystal water was carried out with OPLS force field by hyperchem 7.5 software. In the correlative study, the optimal temperature of wheat monomeric and dimeric protein inhibitors was from 273 K to 318 K. The the average temperature of experimentation is 289 K. (1) The optimal temperature of interaction between alpha-amylase and its inhibitors was 280 K without crystal water that was close to the results of experimentation. The forming of enzyme-water and inhibitor-water was easy, but incorporating third monomer was impossible. (2) Having analyzed the potential energy data, the optimal temperature of interaction energy between alpha-amylase and its inhibitors covering 9 : 1, 5 : 5, 4 : 6, and 1 : 9 proportion crystal water was 290 K. (3) We compared the correlative QSAR properties. The proportion of crystal water was close to the data of polarizability (12.4%) in the QSAR properties. The optimal temperature was 280 K. This result was close to 289 K. These findings have theoretical and practical implications.


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## Body

## 1. Introduction

Alpha-amylases (1, 4-α-D-glucan-4-glucanohydrolase; EC3.  2.1.1)
are endoglycosidases
which catalyze the hydrolysis of internal α-1, 4-D-glucosidic linkages in
starch and dextrins, thereby generating smaller dextrins and oligosaccharides
with a C1–OH group in the
α-anomeric configuration [1].The enzyme is a subset of the alpha-amylases group
of enzymes that are classified as glycosy1 hydrolase family 13 based on amino
acid sequence similarity [2]. Alpha-amylases play a central role in
carbohydrate metabolism of microorganisms, plants, and animals [3]. Furthermore,
they are widely used in food and starch processing industry and, after
proteases, have become the most used enzymes in modern biotechnology [4].Enzyme inhibitors are important tools of nature for regulating the
activity of enzymes in cases of emergency. Plant seeds are known to produce a
variety of enzyme inhibitors that are thought to protect the seed against
insects and microbial pathogens. Proteinase inhibitors are the best studied of
this group [5]; expression of proteinase inhibitor genes in transgenic plants provides
protection against pathogens [6]. Comparatively less is known about the
inhibitors of alpha-amylase which might, on the other hand, be equally
attractive candidates for conferring pest resistance to transgenic plants since
many of them inhibit both proteinases and alpha-amylase. Plants have evolved
defense strategies to counteract these effects through enzyme inhibitors
impeding the action of insect gut digestive alpha-amylases and reducing the postprandial glucose
peaks [7]. Expression of plants inhibitor genes in transgenic plants provides
protection against pathogens (for a review, see Ryan). Comparatively less is
known about the inhibitors of alpha-amylases which might, on the other hand, be
equally attractive candidates for conferring pest resistance to transgenic
plants since many of them inhibit both proteinases and alpha-amylases [8]. The
inhibitor has potentials in various fields, from the treatment of diabetes to
crop protection. Therefore, we are very interested in which mechanisms and
optimal condition of the inhibitor exerted on alpha-amylases.The major alpha-amylases inhibitor (AAI) present in the seeds of
Amaranthus hypochondriacus,
a variety of the Mexican crop plant amaranth, is a 32-residue-long polypeptide
containing 6 cysteines, with 1–4, 2–5, 3–6 three disulfide
bridges. It is the shortest alpha-amylases inhibitor described so far which has
no known close homologs in the sequence data bases [8]. The structural
properties of alpha-amylases have been elucidated by NMR [9]. People are very
interested in which mechanism and optimal condition of inhibitor exerted on
alpha-amylases.Quantitative structure-activity
relationship (QSAR) represents an attempt to relate structural descriptors of
molecules with their physicochemical properties and biological activities. It
is widely used for the prediction of physicochemical properties in chemical, environmental,
and pharmaceutical areas [10, 11]. The main steps implicated in this method
include data collection, molecular descriptor selection and procurement,
correlation model development, and finally model evaluation. At present, many
types of molecular descriptors have been proposed to describe the structural
features of the molecules [12, 13]. The success of QSAR approach can be
explained by the insight offered into the structural determination of chemical
properties, and the possibility to estimate the properties of new chemical
compounds without the need to synthesize and test them [14]. Recently, Anil
Kumar has reported the development of useful QSAR models for antimicrobial activity
[15–17] and anti-inflammatory activity [18].In this work, we
selected the structure of alpha-amylases from Tenebrio molitor larvae
(containing 471 amino acid residues) and inhibitor from the amaranth. A number
of crystal waters were distributed to alpha-amylases and their inhibitors manually
using hyperchem 7.5 software according to different proportion via molecular dynamics
simulation. The Tenebrio molitor alpha-amylase and the amaranth alpha-amylase
inhibitor QSAR properties were calculated. The results showed that the crystal
water had affected in the interaction between the alpha-amylases and their inhibitors. These findings
have theoretical and practical implications.


## 2. Materials and Methods

The structure of Tenebrio molitor alpha-amylase, the amaranth
alpha-amylase inhibitor, and crystal water (containing 273 molecules) was taken
from 1clv (http://www.rcsb.org/pdb/). (1) Alpha-amylase
and inhibitor QSAR properties were calculated out to find out the correlation
using OPLS force field by hyperchem 7.5 software. There are partial charges, surface area
[approx.], surface area [grid], volume, hydration energy, log P, refractivity,
polarizability, and mass of structural variance. (2) The Tenebrio molitor alpha-amylase, the amaranth
alpha-amylase inhibitor, and crystal water were 3 monomers. 3 monomers formed 4 united molecules (enzyme-inhibitor, enzyme-water, inhibitor-
water, and enzyme-inhibitor-water). (3) The partial crystal water formed united molecules with alpha-amylase or inhibitor.
A number of crystal waters were distributed to alpha-amylase and its inhibitor
manually according to different proportion (E:I=9:1,8:2,…,1:9.). The energy of
alpha-amylase, inhibitor, crystal water, and the united molecular structure was
calculated out using OPLS force field by hyperchem 7.5 software. Calculated detail
was the following text.All modeling procedures, including energy minimization and molecular
dynamics, were performed using the hyperchem 7.5 software. Energy calculations
were carried out using the OPLS force field. Optimized molecular structure
until the maximum energy derivative was lower than 0.1 kcal/moL (0.418 kJ/moL)
in order to obtain a correct geometry. Dynamics simulation was performed using
a time step of 0.5femtosecond, and the temperature was altering 10 K from 270 K
to 370 K. There were 3 processes in simulation. Firstly, heating, from 0 K to simulation temperature using 7 K
per step, heating time was 0.1 ps. Secondly, simulating, simulation time was 20
picoseconds in simulation temperature. Finally, annealing, from simulation
temperature to 0 K using 7 K per step, annealing time was
0.1 picosecond [19]. The system was kept for 20.2  picoseconds at each
temperature. After simulation, we collected data of EPOT [20].Recent research showed that enzymes had been used on all conformation not only during catalysis but also
before catalysis. Since the protein motions necessary for catalysis were an
intrinsic property of the enzyme, motion was localized not only to the active
site but also to a wider dynamic network [21]. Thus, it can be seen that molecular state was taken
on all possible conformation during reaction or else process. Therefore, in order to reflect
energy during simulation, we carried out abnegating half potential energy
data of starting simulation and averaging spare potential energy data (twenty
thousand states between 10.1 picoseconds and 20.1 picoseconds). Gained data were
regarded as potential energy at this temperature. We kept enzyme having enough
number of state during the process of simulate temperature and avoided effective influence that system had been arrived at simulate
temperature but was not likely to reach balance at the same time.The energy of
interaction was calculated from experimental data using the following equation [22–24]: (1)ΔE=E−(E1+E2).Here, E was the overall
energy of the binding system; E1 was the energy
of alpha-amylase and crystal water; E2 was the energy of inhibitor and crystal water; ΔE was the interaction energy.


## 3. Results and Discussion

### 3.1. The QSAR Properties

Since predictions from any QSAR models cannot be intrinsically better than
the experimental data employed to develop the model, the quality of the input data
will greatly influence the QSAR model performance. In order to build a QSAR
model with good generalized performance, a preliminary analysis for the quality
of the data set (mainly the detection of outliers) was performed by modeling
the complete set of alpha-amylase and its inhibitor.The QSAR properties of alpha-amylase and its inhibitor were provided in
Table1.Table 1
The QSAR properties of inhibitor and alpha-amylase.
SpeciesQSARInhibitoralpha-amylaseInhibitor/alpha-amylase (%)Partial charges0.000.00—Surface area[approx.]5220.84over—Surface area[grid]8104.7150393.6016.1Volume6860.6043349.1115.8Hydration energy2214.75over—Log P−1.71−1218.300.1Refractivity871.396694.7113.0Polarizability351.612826.7112.4Mass3661.5951193.147.2Because the inhibitor and alpha-amylase were neutral molecules, so their partial
charges were zero in Table1. The proportion of surface area [approx.], hydration
energy of inhibitor, and alpha-amylase was very small and neglected. But the
proportion of surface area [grid], volume, log P, refractivity, polarizability,
and mass were 16.1%, 15.8%, 0.1%, 13.0%, 12.4%, 7.2%, respectively. Therefore, the different distributed
proportion of crystal water was possible close to these QSAR properties
proportion.


### 3.2. Simulate Optimal Temperature among 3 Monomers

We calculated the interaction energy among 3 monomers according to (1). The relation of the
interaction energy with temperature was presented in Figure 1 (the respective chart of ΔE among 3 monomers). From Figure 1,
the interaction energy between alpha-amylase and its inhibitor was negative at
280 K and 290 K, which showed that it was combined and reacted between
alpha-amylase and its inhibitor. However, it was not combined between
alpha-amylase and its inhibitor at the others temperature. The interaction
energy was on the nadir at 280 K which was the optimal temperature between
alpha-amylase and its inhibitor.The respective chart ofΔE among 3 monomers.
(a)(b)(c)(d)(e)(f)This information showed that the interaction energy between crystal water and alpha-amylase inhibitor was negative
from 270 K to 370 K, which showed that they were combined and reacted among
crystal water, alpha-amylase, and its inhibitor. As temperature increases, the
interaction energy between alpha-amylase and its inhibitor tends to get
smaller. However, the interaction energy between crystal water and alpha-amylase inhibitor was negative,
and the numerical value was very big from 270 K to 370 K.The results showed that it was easy to combine between crystal water and alpha-amylase
inhibitor.Analysis of the results indicated that the interaction energy between
alpha-amylase and its inhibitor was negative from 270 K to 370 K. The forming of inhibitor-water was easy, but
the incorporating alpha-amylase was impossible.The interaction energy between alpha-amylase and its inhibitor was negative,
and the numerical value was very big from 270 K to 370 K. The forming of enzyme-water was easy, but the incorporating
inhibitor was
impossible.The result
was as follows: the forming of enzyme-water and inhibitor-water was easy, but the incorporating third monomer was impossible.


### 3.3. Simulate Optimal Temperature at the Different Distributed Proportion of Crystal Water

The interaction energy between alpha-amylase and inhibitor covering
different proportion of crystal water was calculated according to (1). The
relation of the interaction energy with temperature was presented in Figure 2 (the
compositive chart of ΔE in different crystal water proportion) and Figure 3 (the
respective chart of ΔE in different crystal water proportion). Then, we would
discuss them, respectively.Figure 2
The compositive chart of ΔE in different crystal water proportion.The respective chart ofΔE in different crystal water proportion.
(a)(b)(c)(d)(e)(f)(g)(h)(i)From Figure2,
it could be seen that the interaction energy between alpha-amylase and its
inhibitor covering different proportion of crystal water was all negative. This
information showed that it was combined and reacted between alpha-amylase and
its inhibitor from 270 K to 370 K. The interaction energy was on the nadir at
330 K, when the different distributed proportion of crystal water was 2 : 8. In
this condition, the reaction was the easiest between alpha-amylase and its inhibitor.
However, the interaction energy was on the peak at 320 K, when the different
distributed proportion of crystal water was 9 : 1. In this condition, the
reaction was the hardest between alpha-amylase and its inhibitor.From Figure3, the optimal temperature of the interaction between
alpha-amylase and its inhibitor was changed by the distributed proportion of
crystal water. In the correlative study, it was reported that the optimal
temperature of wheat monomeric and dimeric protein inhibitors was from 273 K to
318 K [25, 26].The absolute value of the interaction energy was the greatest at 300 K when
the distributed proportion of crystal water was 9 : 1, 8 : 2, 7 : 3, 5 : 5, 4 : 6, 3 : 7, and
1 : 9. The results showed the optimal temperature via molecular dynamics
simulation which was agreed with the results of experimentation. And the
binding of alpha-amylase average temperature was 289 K.The interaction energy
between alpha-amylase and its inhibitor was on the nadir at 290 K in figure when the distributed proportion of crystal water was
9 : 1, 5 : 5, 4 : 6, and 1 : 9. In the case of 1 : 9, the optimal temperature may be
related to some QSAR properties.The interaction energy between alpha-amylase and its inhibitor was on the
nadir at 340 K, 330 K when the distributed proportion of crystal water was, respectively,
6 : 4 and 2 : 8. These results were disagreeing with the experimental results that
may be caused by the distributed proportion of crystal water and others
causation, which had studied as follow.


### 3.4. Simulate Optimal Temperature in the Case of the Different Distributed Proportion of Crystal Water is 12.4%

For the sake of an accurate result, we must treat jointly the experimental
results related to some QSAR properties. In the case of 1 : 9 (about 11.1%), the
interaction energy between alpha-amylase and its inhibitor was the greatest in
all figures above, and this proportion of crystal water was close to the data of
polarizability (12.4%) in the QSAR properties. This indicated that
polarizability of the QSAR properties possibly had higher influence to the
interaction. We want to validate below that polarizability affected reactive
temperature condition of interaction between alpha-amylase and its inhibitor.We calculated the interaction energy between alpha-amylase and its
inhibitor covering (87.6 : 12.4) proportion of crystal water according to (1).
The relation of the interaction energy with temperature was presented in Figure 4. From Figure 4, the optimal temperature was 280 K which was from 273 K to 318 K.
However, this result was a little different to 289 K (290 k) which was average temperature in the
correlative report. The proportion of Surface Area [Grid], Volume, Refractivity,
and Mass effect in the interaction
between alpha-amylase and its inhibitor will be studied in future.Figure 4
The chart of ΔE in polarizability crystal water proportion.


## 3.1. The QSAR Properties

Since predictions from any QSAR models cannot be intrinsically better than
the experimental data employed to develop the model, the quality of the input data
will greatly influence the QSAR model performance. In order to build a QSAR
model with good generalized performance, a preliminary analysis for the quality
of the data set (mainly the detection of outliers) was performed by modeling
the complete set of alpha-amylase and its inhibitor.The QSAR properties of alpha-amylase and its inhibitor were provided in
Table1.Table 1
The QSAR properties of inhibitor and alpha-amylase.
SpeciesQSARInhibitoralpha-amylaseInhibitor/alpha-amylase (%)Partial charges0.000.00—Surface area[approx.]5220.84over—Surface area[grid]8104.7150393.6016.1Volume6860.6043349.1115.8Hydration energy2214.75over—Log P−1.71−1218.300.1Refractivity871.396694.7113.0Polarizability351.612826.7112.4Mass3661.5951193.147.2Because the inhibitor and alpha-amylase were neutral molecules, so their partial
charges were zero in Table1. The proportion of surface area [approx.], hydration
energy of inhibitor, and alpha-amylase was very small and neglected. But the
proportion of surface area [grid], volume, log P, refractivity, polarizability,
and mass were 16.1%, 15.8%, 0.1%, 13.0%, 12.4%, 7.2%, respectively. Therefore, the different distributed
proportion of crystal water was possible close to these QSAR properties
proportion.


## 3.2. Simulate Optimal Temperature among 3 Monomers

We calculated the interaction energy among 3 monomers according to (1). The relation of the
interaction energy with temperature was presented in Figure 1 (the respective chart of ΔE among 3 monomers). From Figure 1,
the interaction energy between alpha-amylase and its inhibitor was negative at
280 K and 290 K, which showed that it was combined and reacted between
alpha-amylase and its inhibitor. However, it was not combined between
alpha-amylase and its inhibitor at the others temperature. The interaction
energy was on the nadir at 280 K which was the optimal temperature between
alpha-amylase and its inhibitor.The respective chart ofΔE among 3 monomers.
(a)(b)(c)(d)(e)(f)This information showed that the interaction energy between crystal water and alpha-amylase inhibitor was negative
from 270 K to 370 K, which showed that they were combined and reacted among
crystal water, alpha-amylase, and its inhibitor. As temperature increases, the
interaction energy between alpha-amylase and its inhibitor tends to get
smaller. However, the interaction energy between crystal water and alpha-amylase inhibitor was negative,
and the numerical value was very big from 270 K to 370 K.The results showed that it was easy to combine between crystal water and alpha-amylase
inhibitor.Analysis of the results indicated that the interaction energy between
alpha-amylase and its inhibitor was negative from 270 K to 370 K. The forming of inhibitor-water was easy, but
the incorporating alpha-amylase was impossible.The interaction energy between alpha-amylase and its inhibitor was negative,
and the numerical value was very big from 270 K to 370 K. The forming of enzyme-water was easy, but the incorporating
inhibitor was
impossible.The result
was as follows: the forming of enzyme-water and inhibitor-water was easy, but the incorporating third monomer was impossible.


## 3.3. Simulate Optimal Temperature at the Different Distributed Proportion of Crystal Water

The interaction energy between alpha-amylase and inhibitor covering
different proportion of crystal water was calculated according to (1). The
relation of the interaction energy with temperature was presented in Figure 2 (the
compositive chart of ΔE in different crystal water proportion) and Figure 3 (the
respective chart of ΔE in different crystal water proportion). Then, we would
discuss them, respectively.Figure 2
The compositive chart of ΔE in different crystal water proportion.The respective chart ofΔE in different crystal water proportion.
(a)(b)(c)(d)(e)(f)(g)(h)(i)From Figure2,
it could be seen that the interaction energy between alpha-amylase and its
inhibitor covering different proportion of crystal water was all negative. This
information showed that it was combined and reacted between alpha-amylase and
its inhibitor from 270 K to 370 K. The interaction energy was on the nadir at
330 K, when the different distributed proportion of crystal water was 2 : 8. In
this condition, the reaction was the easiest between alpha-amylase and its inhibitor.
However, the interaction energy was on the peak at 320 K, when the different
distributed proportion of crystal water was 9 : 1. In this condition, the
reaction was the hardest between alpha-amylase and its inhibitor.From Figure3, the optimal temperature of the interaction between
alpha-amylase and its inhibitor was changed by the distributed proportion of
crystal water. In the correlative study, it was reported that the optimal
temperature of wheat monomeric and dimeric protein inhibitors was from 273 K to
318 K [25, 26].The absolute value of the interaction energy was the greatest at 300 K when
the distributed proportion of crystal water was 9 : 1, 8 : 2, 7 : 3, 5 : 5, 4 : 6, 3 : 7, and
1 : 9. The results showed the optimal temperature via molecular dynamics
simulation which was agreed with the results of experimentation. And the
binding of alpha-amylase average temperature was 289 K.The interaction energy
between alpha-amylase and its inhibitor was on the nadir at 290 K in figure when the distributed proportion of crystal water was
9 : 1, 5 : 5, 4 : 6, and 1 : 9. In the case of 1 : 9, the optimal temperature may be
related to some QSAR properties.The interaction energy between alpha-amylase and its inhibitor was on the
nadir at 340 K, 330 K when the distributed proportion of crystal water was, respectively,
6 : 4 and 2 : 8. These results were disagreeing with the experimental results that
may be caused by the distributed proportion of crystal water and others
causation, which had studied as follow.


## 3.4. Simulate Optimal Temperature in the Case of the Different Distributed Proportion of Crystal Water is 12.4%

For the sake of an accurate result, we must treat jointly the experimental
results related to some QSAR properties. In the case of 1 : 9 (about 11.1%), the
interaction energy between alpha-amylase and its inhibitor was the greatest in
all figures above, and this proportion of crystal water was close to the data of
polarizability (12.4%) in the QSAR properties. This indicated that
polarizability of the QSAR properties possibly had higher influence to the
interaction. We want to validate below that polarizability affected reactive
temperature condition of interaction between alpha-amylase and its inhibitor.We calculated the interaction energy between alpha-amylase and its
inhibitor covering (87.6 : 12.4) proportion of crystal water according to (1).
The relation of the interaction energy with temperature was presented in Figure 4. From Figure 4, the optimal temperature was 280 K which was from 273 K to 318 K.
However, this result was a little different to 289 K (290 k) which was average temperature in the
correlative report. The proportion of Surface Area [Grid], Volume, Refractivity,
and Mass effect in the interaction
between alpha-amylase and its inhibitor will be studied in future.Figure 4
The chart of ΔE in polarizability crystal water proportion.



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*Source: 469062-2008-05-11.xml*