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Noncovalent PEGylation Through Protein-Polyelectrolyte Interaction: Kinetic Experiment and Molecular Dynamics Simulation Takaaki Kurinomaru, Kengo Kuwada, Shunsuke Tomita, Tomoshi Kameda, and Kentaro Shiraki J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b02741 • Publication Date (Web): 26 Jun 2017 Downloaded from http://pubs.acs.org on June 27, 2017

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The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

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Noncovalent

PEGylation

through

Protein-

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Polyelectrolyte Interaction: Kinetic Experiment and

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Molecular Dynamics Simulation

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Takaaki Kurinomaru a, Kengo Kuwada b, Shunsuke Tomita a, Tomoshi Kameda c, and Kentaro

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Shiraki b *

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a

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(AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology

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b

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305-8573, Japan

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c

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Technology (AIST), 2-4-7 Aomi, Koto, Tokyo 135-0064, Japan

Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki

Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and

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* To whom correspondence should be addressed. Tel: +81-29-8535306. Fax: +81-29-8535215. E-

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mail: [email protected]

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Abstract

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Noncovalent binding of polyethylene glycol (PEG) to a protein surface is a unique protein

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handling technique to control protein function and stability. A diblock copolymer containing PEG

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and polyelectrolyte chains (PEGylated polyelectrolyte) is a promising candidate for noncovalent

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attachment of PEG to a protein surface because of the binding through multiple electrostatic

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interactions without protein denaturation. To obtain a deeper understanding of protein–

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polyelectrolyte interaction at the molecular level, we investigated the manner in which cationic

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PEGylated polyelectrolyte binds to anionic α-amylase in enzyme kinetic experiments and molecular

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dynamics (MD) simulations. Cationic PEG-block-poly(N,N-dimethylaminoethyl) (PEG-b-PAMA)

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inhibited the enzyme activity of anionic α-amylase due to binding of PAMA chains. Enzyme

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kinetics revealed that the inhibition of α-amylase activity by PEG-b-PAMA is noncompetitive

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inhibition manner. In MD simulations, the PEG-b-PAMA molecule was initially located at six

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different placements of the x-, y-, and z-axis ± 20 Å from the center of α-amylase, which showed

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that the PEG-b-PAMA nonspecifically bound to the α-amylase surface, corresponding to the

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noncompetitive inhibition manner that stems from the polymer binding to enzyme surface other than

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the active site. In addition, the enzyme activity of α-amylase in the presence of PEG-b-PAMA was

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not inhibited by increasing the ionic strength, consistent with the MD simulation, i.e., PEG-b-PAMA

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did not interact with α-amylase in high ionic strength conditions. The results reported in this paper

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suggest that enzyme inhibition by PEGylated polyelectrolyte can be attributed to the random

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electrostatic interaction between protein and polyelectrolyte.

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Introduction

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PEGylation is a technique for modification of proteins, peptides, and other polymers by

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polyethylene glycol (PEG). PEGylation has been used extensively in the biomedical field to increase

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the circulation half-life, structural stability, and shelf-life of therapeutic proteins.1–4 Since early

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examples reported by Abuchowski et al. in the 1970’s,5,6 several strategies have been proposed for

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protein PEGylation.7 These strategies are classified simply as covalent or noncovalent approaches.

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At present, almost all of the commonly used PEGylation strategies involve covalent approaches, and

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12 types of PEGylated protein are commercially available for various diseases, including severe

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combined immunodeficiency disease, acute lymphoblastic leukemia, and refractory chronic gout.8

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However, covalent PEGylation requires complex procedures, including chemical reaction for

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covalent attachment of PEG chains to the protein of interest. The PEGs bound to the protein often

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reduce the biological activity.9–11 In contrast, noncovalent PEGylation is a promising method that do

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not require any chemical modification or mutagenesis of the protein of interest, and therefore can

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minimize the undesired stress on the protein structure.12 Generally, polymers for noncovalent

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PEGylation of protein consist of PEG segments and key functional segments that can attach to

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proteins, such as hydrophobic groups,13–19 sugars,20,21 and metal-binding groups.22

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We reported previously that the diblock copolymer containing PEG and polyelectrolyte, so-

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called PEGylated polyelectrolyte, stabilized therapeutic protein in vitro by noncovalent

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PEGylation.23 The polyelectrolyte chains of the copolymer interact with complementary charged

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proteins, whereas the PEG chains contribute to a stabilization effect due to its strong hydration and

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high

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dimethylaminoethyl) (PEG-b-PAMA) and anionic L-asparaginase formed a water-soluble complex,

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which retained not only the original enzyme activity and secondary structure of the protein but also

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protected the protein against protease digestion and shaking-induced aggregation.23 Similarly,

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cationic PEG-b-PAMA derivatives bound to anionic α-amylase and β-galactosidase with formation

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of a water-soluble complex.24,25

degree

of conformational flexibility. For example, cationic

PEG-block-poly(N,N-



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Another key aspect is that noncovalent PEGylation through electrostatic interactions enables

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reversible on/off switching of enzyme activity for certain types of protein.24,25 Briefly, the formation

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of a water-soluble complex between protein and PEGylated polyelectrolyte results in complete

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inhibition of the enzyme activity; the subsequent addition of other electrolytes, typically salts or

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oppositely charged polyelectrolytes, results in recovery of the enzyme activity by dissociating the

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bound PEGylated polyelectrolyte from the protein surface. For example, an anionic enzyme is

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inactivated by the addition of cationic PEG-b-PAMA derivatives due to wrapping through

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electrostatic interaction, followed by reactivation of the enzyme activity by addition of anionic

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poly(acrylic acid).24,25 Due to the high degree of reversibility, enzymes inhibited through water-

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soluble complex formation with PEGylated polyelectrolyte have been applied as sensing elements in

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cross-reactive biosensors.26–28 These facile, noncovalent, and reversible modifications of protein

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characteristics have recently been named “wrap-and-strip technology,” and are expected to be useful

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for biopharmaceutical applications to allow regulation of protein function and stability at will in

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living organisms as well as in vitro.29

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Although protein–polyelectrolyte complex formation is mainly driven by electrostatic

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interactions, the manner of binding to the enzyme by polyelectrolyte remains unclear at the atomic

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level. Here, we investigated enzyme inhibition by PEGylated polyelectrolyte using enzyme kinetics

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experiments and molecular dynamics (MD) simulation. As expected, the anionic α-amylase was

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inactivated by addition of cationic PEG-b-PAMA. Enzyme kinetics studies showed that PEG-b-

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PAMA is a noncompetitive inhibitor of α-amylase, indicating that PEG-b-PAMA did not bind

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specifically to the active site of enzyme. The important finding was that the nonspecific interactions

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between PEG-b-PAMA and α-amylase were observed by MD simulation. The PEG-b-PAMA did

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not bind to α-amylase by increasing ionic strength as demonstrated by inhibition experiments and

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MD simulation. We concluded that the random electrostatic interaction of PEGylated polyelectrolyte

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caused reversible inactivation of the α-amylase.


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Experimental Section

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Materials

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Aspergillus oryzae α-amylase and 3-(N-morpholino) propanesulfonic acid (MOPS) were

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from Sigma Chemical. Co. (St. Louis, MO). 2-Chloro-4-nitrophenyl-α-D-maltotrioside was from

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Wako Pure Chemical Ind., Ltd. (Osaka, Japan). Sodium chloride (NaCl) was from Nacalai Tesque

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(Kyoto, Japan). These chemicals used were of high quality analytical grade and were used as

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received. Polyethylene glycol-block-poly(N,N-dimethylaminoethyl methacrylate) (PEG-b-PAMA)

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with an average molecular weight of 10.0 kDa (PEG chains, 4.5 kDa; PAMA chains, 5.5 kDa) was

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synthesized as reported previously.27

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Protein concentrations

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The protein concentrations of α-amylase were determined from the absorbance at 280 nm

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using a spectrophotometer (V-630; Japan Spectroscopic Company, Ltd., Tokyo, Japan), with

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extinction coefficient of 106,160 M–1 cm–1.30

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Inhibition of the α-amylase activity by PEG-b-PAMA

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Solutions of 1.0 µM α-amylase and 0 – 5.0 µM PEG-b-PAMA in 10 mM MOPS buffer

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containing 0 – 150 mM NaCl (pH 7.0) were prepared and incubated for 2 hours at 25°C. After

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incubation, α-amylase enzyme activity was measured by colorimetric enzyme assay as described

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below.

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Enzyme assay A solution containing of 150 µL containing 0 – 60 mM substrate (2-chloro-4-nitrophenyl-α-

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D-maltotrioside)

in 10 mM MOPS buffer (pH 7.0) was mixed with 150 µL of enzyme solution. The

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initial reaction rate (v0) was determined from the slope of the initial increase in the intensity of

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absorbance at 410 nm for 60 s using a spectrophotometer (V-630; Japan Spectroscopic Company, 5 ACS Paragon Plus Environment


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Ltd.). The absorbance was converted to concentration using a molecular extinction coefficient for 2-

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chloro-4-nitrophenol of 15200 M-1cm-1. Enzyme activity was defined as the ratio of v0 in the

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presence of polyelectrolyte to that with no polyelectrolyte. For the salt effect studies, all parameters

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were unchanged except for the concentration of NaCl. The average of three independent

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measurements under the same conditions was used as the enzyme activity of α-amylase. The activity

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of α-amylase in the presence of the polyelectrolytes was normalized to that without polyelectrolytes

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at the same salt concentration because the v0 of α-amylase changed slightly with increasing NaCl

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concentration.

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Enzyme kinetic analysis

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Enzyme kinetics analysis was performed according to classical methods.31 Briefly, the

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Michaelis constant (KM) and the catalytic constant (kcat) of α-amylase were calculated by linear

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fitting of the 1/v0 – 1/[S] plot (Figure 2B) of the Lineweaver–Burk equation:

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(1)

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where [E] and [S] are enzyme and substrate concentrations, respectively.

133 134

Molecular dynamics simulation of α-amylase in the presence of PEG-b-PAMA

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The microscopic states of the interaction between protein and PEGylated polyelectrolyte

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were studied using MD simulations. The 10 ns calculations were performed twice for one α-amylase

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molecule and one polymer molecule solution with 0 or 0.15 M NaCl. The duration of MD simulation

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was sufficient to the protein/polyelectrolyte interaction and the conformational changes of the

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protein itself. MD simulations started from six initial conformations of the polymer. In initial

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conformations, the polymer molecule was located at six different placements of the x-, y-, and z-axis

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± 20 Å by the α-amylase molecule, and thus the total number of simulations was 12 for each 6 ACS Paragon Plus Environment

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solution. The α-amylase molecules were described using the AMBER ff99SB force field.32 The

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PEG-b-PAMA molecule was described using the general AMBER force field (GAFF).33 A

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restrained electrostatic potential (RESP) change was used for the PEG-b-PAMA molecules.34 The

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solvation and ionic effect were modeled by the Generalized Born (GB) energy.35 The simulations

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were conducted with the NVT ensemble (298 K). The temperature was controlled using a Langevin

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thermostat. To accelerate dynamics, we set the solvent viscosity of water to 1.0 ps-1 (the value of

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normal viscosity of water is 91 ps-1). Pande V and coworker showed the folding time of peptide from

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extended to native conformation acquired by Langevin dynamics simulation with GB implicit

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solvent and 1.0 ps-1 viscosity is about 1/10.36 Thus, the calculation time of 10 ns corresponds to

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about 100 ns practically, which is enough to equilibrate the protein-polymer interaction. The

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noncovalent interactions, such as electrostatics, van der Waals, and Generalized Born, were used

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without cutoffs.37 The covalent bonds of hydrogen atoms in α-amylase and polyelectrolyte were

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constrained using the SHAKE method38 and the integration time step was 2 fs. The simulations were

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conducted using the Amber12 simulator.39 Images were generated in PyMOL (Ver. 1.7 Schrödinger,

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LLC).



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Results

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Inactivation of α-amylase by the binding of PEG-b-PAMA

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The experimental conditions were as follows. α-Amylase from Aspergillus oryzae (Mw: 52.4

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kDa, pI = 4.2) was selected as the model enzyme (Figure 1A). It has been reported that the α-

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amylase enzyme activity can be regulated by a cationic polyelectrolyte due to protein–

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polyelectrolyte complex formation.24–26,28,40 As this enzyme hydrolyzes α-D-(1-4) glycoside bonds of

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oligosaccharides, we selected 2-chloro-4-nitrophenyl-α-D-maltotrioside as a substrate to detect the

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enzyme activity of α-amylase.

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To confirm the inhibitory behavior, the α-amylase enzyme activity was investigated in the

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presence of PEG-b-PAMA (Figure 1B). As expected, the enzyme activity of α-amylase decreased

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with increasing concentration of PEG-b-PAMA: 0.5 µM α-amylase was fully inactivated in the

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presence of 1.5 µM (3 equiv.) PEG-b-PAMA (Figure 2A), coincident with the previous result.24,25

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These results indicated that cationic PEG-b-PAMA binds to the anionic α-amylase surface and then

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inactivated the enzyme.

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Enzyme kinetics experiments were then performed using various concentrations of PEG-b-

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PAMA to determine the manner of inhibition. Figure 2B shows the Lineweaver–Burk plot for the

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hydrolysis of substrate by α-amylase in the absence and presence of PEG-b-PAMA. The Michaelis

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constant (KM) and catalytic constant (kcat) were calculated by linear fitting to the Lineweaver–Burk

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equation (eq. 1) for each PEG-b-PAMA concentration (Table 1). The addition of PEG-b-PAMA to

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α-amylase decreased kcat with increasing concentration of PEG-b-PAMA. On the other hand, KM

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remained unchanged by addition of 0.5 equiv. PEG-b-PAMA, but increased for 1.0 equiv. These

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results suggested that PEG-b-PAMA showed typical noncompetitive inhibition (Figure 2C).

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Molecular dynamics simulation of interaction between α-amylase and PEG-b-PAMA

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To understand the binding behavior at atomic level resolution, we conducted MD simulations

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of the α-amylase/PEG-b-PAMA system. In this system, water and ions were not included explicitly, 8 ACS Paragon Plus Environment

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but the solvation and ionic effect were represented by the Generalized Born (GB) energy known as

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the implicit solvent.35 MD simulation was performed with one α-amylase molecule and one PEG-b-

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PAMA molecule in water for 10 ns. Note that PEG-b-PAMA was initially located at the six initial

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placements of xyz-axis ± 20 Å by central α-amylase, and thus the total number of simulations was 12

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for each solution condition. Figure 3A shows overlaid representative snapshots of α-amylase with

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PEG-b-PAMA without ionic effects. PEG-b-PAMA bound onto α-amylase molecules within 10 ns,

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but the binding sites on α-amylase were different depending on the relative configuration of PEG-b-

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PAMA to α-amylase at beginning of calculation (see details in Figure S1). It was noted that PEG-b-

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PAMA did not bind to the active site of the enzyme directly, coincident with the noncompetitive

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inhibition of the enzyme by PEGylated polyelectrolyte (Figure 2C). Moreover, the tertiary structure

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of the protein was not altered in any of the simulations, suggesting that the nonspecific binding of

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PEGylated polyelectrolyte contributed to the noncompetitive inhibition of α-amylase enzyme

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activity, rather than inactivation resulting from denaturation.

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Additional MD simulation with the ionic effect was conducted to investigate what governs

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the binding behavior of polyelectrolytes to proteins. We simulated the α-amylase/PEG-b-PAMA

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system with the ionic effect corresponding to 0.15 M NaCl. In contrast to the case without the ionic

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effect, the MD simulation showed that PEG-b-PAMA did not bind to the protein surface, regardless

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of the initial placement of PEG-b-PAMA (Figure 3B). It was not unexpected that the data indicated

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that electrostatic interactions between proteins and polyelectrolytes are shielded by increases in ionic

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strength (typically > 50 mM), resulting in suppression of protein–polyelectrolyte complex

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formation.41,42 Figure S2 shows each energy average (E) and difference of energy between the initial

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coordinate and the coordinate after 10 ns simulation (∆E). There is significant distinction of ∆E

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between α-amylase/PEG-b-PAMA system with 0.15 M NaCl and without ionic effect, suggesting

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that the both electrostatic interaction and solvation effect play important role in protein-polymer

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binding. In addition, the enzyme activity of α-amylase inactivated by PEG-b-PAMA recovered

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rapidly with increasing concentration of NaCl above 50 mM (Figure S3). These results indicated 9 ACS Paragon Plus Environment


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that the electrostatic interaction is a predominant driving force for nonspecific binding of the

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polyelectrolyte to the protein.

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The above MD simulation showed that the binding of PEG-b-PAMA on the protein surface

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was not specific, i.e., binding did not occur at the active site. PEG-b-PAMA molecules have three

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types of binding position: (i) tertiary ammonium groups of PAMA, (ii) ester groups of PAMA, and

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(iii) ether groups of PEG. Figure 4 shows the detailed mapping of major binding sites of PEG-b-

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PAMA on the surface of α-amylase. It is clear that the number of amino acid residues binding to

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tertiary ammonium groups of PAMA was greater than those binding to esters of PAMA and ethers

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of PEG, except for the case that PEG-b-PAMA was initially placed at –20 Å (z-axis) (Figure 4F).

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These results indicated that the tertiary ammonium groups of PAMA primarily contributed to the

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noncovalent interaction between protein and polyelectrolyte. In addition, glutamic acid and aspartic

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acid residues were mainly located close to the tertiary ammonium groups of PAMA, which was also

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caused by the electrostatic interaction between tertiary ammonium groups of PAMA and acidic

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residues of the protein. Furthermore, these mappings revealed that the binding sites of PEG-b-

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PAMA were clearly different depending on the initial placement, suggesting that there were multiple

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polyelectrolyte binding patterns.


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Discussion

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Enzyme inhibitors are typically classified into two types—competitive inhibitors occupy the

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active site in the enzyme thus preventing substrate access, while noncompetitive inhibitors bind at

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enzyme surface that are different from the active site. We previously demonstrated that anionic α-

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amylase and cationic PEG-b-PAMA formed the water-soluble complex using dynamic light

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scattering,24,25 although the binding manner was unclear. In addition, the secondary structure of α-

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amylase did not change with binding by PEG-b-PAMA derivatives.24,25 This study showed that

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PEG-b-PAMA bound to the α-amylase surface, and acted as a noncompetitive inhibitor of enzyme

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activity (Figure 2). The binding behavior was consistent with the results of MD simulation; PEG-b-

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PAMA tended to bind on the enzyme surface other than the active site (Figure 3A). Binding maps

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also showed that there were multiple binding patterns of PEG-b-PAMA (Figure 4). Due to the non-

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homogeneity of the charge distribution of the protein surface, the diversity of binding pattern is a

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common feature of protein–polyelectrolyte interaction. The interactions may involve modification of

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the charge distribution of the enzyme surface following electrostatic binding of polyelectrolyte,

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resulting in the inhibition of enzyme–substrate interaction. In addition, enzyme kinetic data revealed

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that PEG-b-PAMA increased the KM (Table 1), indicating that the polyelectrolytes was related to

241

weaken the affinity between the enzyme and the substrate. Taken together, the enzyme inhibition

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using PEG-b-PAMA was due to nonspecific binding of polyelectrolyte to the enzyme surface,

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possibly resulting in preventing association of the enzyme and substrate or conversion from substrate

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to product.

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Charged regions of a polyelectrolyte molecule can generally interact with complementarily

246

charged or hydrophilic regions on protein surfaces, thus leading to noncovalent complex formation

247

by electrostatic or hydrogen bonds. Accordingly, the electrostatic pairs of tertiary ammonium groups

248

of PAMA with acidic residues of the protein are thought to play significant roles in protein–

249

polyelectrolyte complex formation. In fact, MD simulation in this study revealed that PEG-b-PAMA

250

did not bind to α-amylase in the presence of 0.15 M NaCl, which suppresses electrostatic

251

interactions between complementary charged pairs (Figure 3B). In addition, the binding map in 11 ACS Paragon Plus Environment


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Figure 4 shows that the amino groups of PAMA are prone to interact with several anionic residues,

253

including aspartic acid and glutamic acid. Therefore, it was concluded that the noncovalent binding

254

of PEG-b-PAMA to α-amylase could be attributed to the electrostatic interaction between α-

255

amylase and PAMA.

256

MD simulation revealed the interesting behavior in that the PEG chain, as well as PAMA, of

257

PEG-b-PAMA binds to α-amylase (Figure 4), while α-amylase enzyme activity remained constant

258

with the addition of PEG (data not shown). PEG is known as a biologically inert polymer which do

259

not interact to protein molecules except when used at high concentration.43 It was suggested that the

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electrostatically bound PAMA chain brought the PEG chain of PEG-b-PAMA close to the α-

261

amylase surface, facilitating the nonspecific interaction between PEG and protein. Accordingly,

262

binding of the PAMA chain to the protein surface caused subsequent binding of the PEG chain.

263

Electrostatic interaction-based noncovalent PEGylation is typically salt-responsive because

264

distribution of Coulomb potential around a protein is modulated depending on the ionic strength.44

265

The increase in ionic strength hampers the electrostatic interaction between protein and

266

polyelectrolyte due to electrostatic shielding. In general, the physiological environment contains 15

267

– 150 mM ions, which results in release of the protein from PEGylated polyelectrolyte.45 To address

268

this issue, we have designed a functionalized PEG-b-PAMA with hydrophobic groups, which

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successfully form complex with α-amylase activity even in the presence of 50 mM NaCl.25 The

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result indicates that both charged and hydrophobic groups play indispensable roles in the formation

271

of the stable protein-polyelectrolyte complex under physiological salt concentrations. For use of

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noncovalent PEGylation in biomedical applications, it will be important to design functional

273

polyelectrolytes with high affinity for protein under conditions of physiological ionic strength.

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Prediction of polyelectrolyte behaviors in response to ionic strength increase using MD simulation

275

would help to propose design guidelines for the PEGylated polyelectrolyte.


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Conclusion

277

In summary, we investigated the interaction between protein and PEGylated polyelectrolyte

278

by a combination of experimental and computational approaches. Cationic PEG-b-PAMA bound

279

nonspecifically to the α-amylase surface, resulting in noncompetitive inhibition of the enzyme. In

280

addition, the ionic effect suppressed the electrostatic binding between α-amylase and PEG-b-PAMA

281

observed by MD simulation, whereby the α-amylase activity did not change. To our knowledge, this

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is the first study in which MD simulations were used to predict the binding behavior between protein

283

and PEGylated polyelectrolyte. Insight into the nonspecific interactions between protein and

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PEGylated polyelectrolyte will be indispensable for understanding the mechanism of not only

285

manipulation of enzyme activity but also stabilization of pharmaceutical protein by noncovalent

286

PEGylation. We believe that the noncovalent PEGylation technologies will expand the applications

287

of therapeutic proteins, such as stabilization during storage and drug delivery systems.

288 289

Associated Content

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The Supporting Information is available free of charge on the ACS Publications website.

291

Three figures showing Representative snapshots protein-polymer binding calculated by MD

292

simulation, comparison of the potential energy of the α-amylase/PEG-b-PAMA system, effect of

293

ionic strength of the enzyme activity (PDF)

294 295

Acknowledgments

296

The theoretical calculations were performed using the Research Center for Computational

297

Science, Okazaki, Japan, and super computer system, National Institute of Genetics (NIG), Research

298

Organization of Information and Systems (ROIS). This study was supported in part by University of

299

Tsukuba and JSPS KAKENHI (Grant Numbers 15K13812 and 15H03583) from the Ministry of

300

Education, Culture, Sports, Science, and Technology (MEXT), Japan.

301 13 ACS Paragon Plus Environment


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Table Legends

420

Table 1. Enzyme kinetic parameters α-amylase in the presence of PEG-b-PAMA kcat [a] (s-1)

KM [a] (mM)

0 eq.

6.6

23.2

0.5 eq.

3.6

20.8

1.0 eq.

2.5

57.4

Molar ratio

421 422

[a]

Calculated by linear fitting of Figure 2B.

423



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424

Figure Legends

425

Figure 1. (A) Crystal structure of α-amylase (PDB: 6TAA). The active site is shown by green

426

spheres. (B) Chemical structure of PEG-b-PAMA.

427

Figure 2. (A) Inhibition of anionic α-amylase in the presence PEG-b-PAMA. The normalized

428

enzyme activities of 0.5 µM α-amylase for 20 mM substrate with various concentrations of PEG-b-

429

PAMA were measured in 10 mM MOPS buffer (pH 7.0). The curve was added to guide the eye. (B)

430

Lineweaver–Burk plots for hydrolysis of substrate by 0.5 µM α-amylase (closed circles) in the

431

presence of 0.5 equiv. (open circles) and 1.0 equiv. (closed squares) PEG-b-PAMA in MOPS buffer

432

(pH 7.0). The lines represent the best fit for the data using eq. 1. (C) Schematic illustration of

433

noncompetitive inhibition of α-amylase activity by PEG-b-PAMA. E, enzyme; S, substrate; I,

434

inhibitor; ES; enzyme-substrate complex; EI, enzyme-inhibitor complex; EIS, enzyme-inhibitor-

435

substrate complex; P, product.

436

Figure 3. Overlaid representative snapshots of PEG-b-PAMA binding to the α-amylase surface in

437

the absence (A) or presence (B) of ionic effect (equivalent to 0.15 M NaCl) calculated by MD

438

simulation. Active site residues and other regions are represented as green spheres and gray cartoons,

439

respectively. PEG and PAMA in PEG-b-PAMA are colored purple and cyan, respectively. The

440

binding site of the enzyme with PEG-b-PAMA is shown in yellow.

441

Figure 4. The binding map of PEG-b-PAMA onto the surface of α-amylase calculated by MD

442

simulation. The molecular surface was generated in PyMOL. The colored residues are shown in the

443

binding surfaces of α-amylase to PEG-b-PAMA within a distance of 2.7 Å from tertiary ammonium

444

groups of PAMA (red), ester groups of PAMA (blue), and ether groups of PEG (purple). The active

445

site of α-amylase is shown in green. Initial placements of PEG-b-PAMA were x-axis direction +20

446

Å (A) and –20 Å (B), y-axis direction +20 Å (C) and –20 Å (D), and z-axis direction +20 Å (E) and

447

–20 Å (F) from the center of protein molecule.

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Table of contents

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Figure 1. (A) Crystal structure of α-amylase (PDB: 6TAA). The active site is shown by green spheres. (B) Chemical structure of PEG-b-PAMA. 82x41mm (300 x 300 DPI)

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Figure 2. (A) Inhibition of anionic α-amylase in the presence PEG-b-PAMA. The normalized enzyme activities of 0.5 µM α-amylase for 20 mM substrate with various concentrations of PEG-b-PAMA were measured in 10 mM MOPS buffer (pH 7.0). The curve was added to guide the eye. (B) Lineweaver–Burk plots for hydrolysis of substrate by 0.5 µM α-amylase (closed circles) in the presence of 0.5 equiv. (open circles) and 1.0 equiv. (closed squares) PEG-b-PAMA in MOPS buffer (pH 7.0). The lines represent the best fit for the data using eq. 1. (C) Schematic illustration of noncompetitive inhibition of α-amylase activity by PEG-b-PAMA. E, enzyme; S, substrate; I, inhibitor; ES; enzyme-substrate complex; EI, enzyme-inhibitor complex; EIS, enzyme-inhibitor-substrate complex; P, product. 82x95mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Overlaid representative snapshots of PEG-b-PAMA binding to the α-amylase surface in the absence (A) or presence (B) of ionic effect (equivalent to 0.15 M NaCl) calculated by MD simulation. Active site residues and other regions are represented as green spheres and gray cartoons, respectively. PEG and PAMA in PEG-b-PAMA are colored purple and cyan, respectively. The binding site of the enzyme with PEG-bPAMA is shown in yellow. 177x98mm (300 x 300 DPI)


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Figure 4. The binding map of PEG-b-PAMA onto the surface of α-amylase calculated by MD simulation. The molecular surface was generated in PyMOL. The colored residues are shown in the binding surfaces of αamylase to PEG-b-PAMA within a distance of 2.7 Å from tertiary ammonium groups of PAMA (red), ester groups of PAMA (blue), and ether groups of PEG (purple). The active site of α-amylase is shown in green. Initial placements of PEG-b-PAMA were x-axis direction +20 Å (A) and –20 Å (B), y-axis direction +20 Å (C) and –20 Å (D), and z-axis direction +20 Å (E) and –20 Å (F) from the center of protein molecule. 177x104mm (300 x 300 DPI)


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