From the Department of Life Science,
§ Institute of Biochemistry, National Yang-Ming University,
Taipei 11221, Taiwan, ¶ Department of Medical Research and
Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan,
Institute of Biological Chemistry, Academia Sinica,
Taipei 11529, Taiwan, ** Department of Biochemistry,
National Taipei College of Nursing, Taipei 11219, Taiwan, and
X-ray Structural Biology Group,
Synchrotron Radiation Research Center, Hsinchu
30077, Taiwan
Received for publication, October 22, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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D-Aminoacylase is an attractive
candidate for commercial production of D-amino acids
through its catalysis in the hydrolysis of
N-acyl-D-amino acids. We report here the
first D-aminoacylase crystal structure from A. faecalis at 1.5-Å resolution. The protein comprises a small
N-Acyl-D-amino acid amidohydrolases
(D-aminoacylases, EC 3.5.1.14) catalyze the zinc-assisted
hydrolysis of N-acyl-D- amino acids to produce
the corresponding D-amino acids, which are intermediates in
the preparation of pesticides, bioactive peptides, and antibiotics. Recently, D-amino acids have been found in bacteria,
plants, and animals, and their physiological functions have received
increased attention. Production of L-amino acids by optical
resolution using L-aminoacylase immobilized on
DEAE-Sephadex has been used in industry. Therefore, production of
D-amino acids using D-aminoacylase has commercial importance.
Several D-aminoacylases screened from microorganisms in
various soils have been isolated and characterized (1-6). Because of
more thermostability, high substrate specificity with hydrophobic D-amino acids such as
N-acetyl-D-methionine, and high affinity to DEAE
resins, the D-aminoacylase from Alcaligenes
faecalis DA1 is more suitable for optical resolution of
N-acyl-DL-amino acids (2). The DA1
D-aminoacylase shares 40-80% sequence identity to those
from A. xylosoxydans A-6, and Pyrococcus abyssi,
but no significant homology with L-aminoacylases (7-10).
Sequence homology search also revealed that the enzyme N-terminal
segment (residues 8-96) shared significant similarity within a variety of amidohydrolases including urease (10). The structural fold was
predicted to be similar to urease and dihydroorotase, which have
grouped into a novel The high degree of global structure and the metal center similarity of
phosphotriestase, adenosine deaminase, and urease have been noted once
these structures were solved (13). Subsequent superposition of these
three protein structures by Holm and Sander (11) revealed a common
ellipsoidal ( On the basis of the metal centers in the known crystal structures, the
superfamily has been divided into two subsets: urease (13),
phosphotriesterase (14), phosphotriesterase homology protein (15),
dihydroorotase (16), and dihydropyrimidinase (17), containing a
binuclear center; and adenosine deaminase (18) and cytosine deaminase
(19) with a mononuclear center (see below). We report here that
D-aminoacylase can be described as a defining member of a
novel subset based on its unusual metal center. The enzyme structure
also suggests the substrate specificity and the catalytic mechanism.
The recombinant protein was expressed, isolated, and
crystallized as described previously (20). All x-ray data were
collected at 100 K. The crystals belong to space group
P212121, with cell dimensions
a = 60.2 Å, b = 76.6 Å, and
c = 135.3 Å. The structure was solved using the Se-SAD
methods (20) and was then refined by using CNS (21). The x-ray data
were collected at beamlines BL6A and BL18B at the Photon Factory,
Tsukuba, Japan, and BL41XU and BL12B2 at SPring-8, Sayo, Japan.
The refinement parameters are presented in Table
I. More than 91% of the
residues are in the most favored regions, with the remaining ones in
the additional allowed regions except His250,
Thr290, and Thr406 due to hydrogen bond
interactions. Figs. 1, 3B, and 4 were generated by MOLSCRIPT
(22), Fig. 2 by INSIGHT II (Molecular Simulation Inc.), Fig.
3A by BOBSCRIPT (23), and Fig. 5 by GRASP (24). The atomic
coordinates and structure factors have been deposited in the Protein
Data Bank (code 1M7J).
The Overall Structure--
The DA1 D-aminoacylase has
483 amino acids, and the current model contains residues 7-480 with
clear electron density. The protein is composed of a small
Structural similarity search by DALI (25) revealed that the closest
structural matches of D-aminoacylase are seven members of
the recently identified A Mononuclear Metalloenzyme with a Binuclear Active
Site--
Unexpectedly, the active site contains only one tightly
bound metal ion (Fig. 3A). On
the basis of the atomic absorption analysis (10) and the zinc anomalous
data, this metal ion is assigned as zinc. The zinc ion is tetrahedrally
coordinated by Cys96 S
There is another potential metal-binding site, surrounded by
His67, His69, Cys96,
Asp366, and ACT1, and similar to the metal-binding sites in
the
Thus, the DA1 D-aminoacylase binds two zinc ions with
widely different affinities. Only the tightly bound zinc is required for the enzyme activity, because the isolated enzyme exhibits significant activity. Addition of extrinsic zinc ions does not enhance
the enzyme activity. A large excess of zinc ions even strongly inhibits
the enzyme activity (data not shown). Therefore, the DA1
D-aminoacylase is a mononuclear enzyme but contains a binuclear active site, bearing an interesting analogy to the
Even though A Novel Subset of the
In D-aminoacylase, one zinc ion binds strongly at the
Structural superposition demonstrates that the metal centers in the
same subset are virtually identical (10, 17, 18). Remarkably,
superposition of the metal centers in different subsets, i.e. D-aminoacylse, urease, and cytosine
deaminase, reveals that the metal ligands are also at the similar
spatial positions, with Cys96 occupying the position of the
carboxylated lysine (Fig. 3B). In cytosine deaminase and
adenosine deaminase, the third conserved histidine compensates the
missing carboxylated lysine. Approximately two-thirds
phosphotriesterase homology proteins such as those from human, mouse,
rat, fly, Bacillus, Salmonella, and
Escherichia coli, use a glutamate instead of the
carboxylated lysine, resulting a larger structural difference at the
The Putative Substrate-binding Site--
Two acetate molecules
from the crystallization solution are observed in the active site
region (Fig. 4A). The first
one (ACT1) ligating the zinc ion(s) may occupy the product
acetate-binding site in the hydrolysis of
N-acteyl-D-amino acid. The second one (ACT2)
forms extensive interactions with Lys252 N
On the basis of three assumptions, the preferred substrate
N-acetyl-D-methionine was modeled into the
active site (Fig. 4B). First, the carboxylate group occupies
the position of that of ACT2 because of the extensive interactions
mentioned above. Second, the side chain binds at the hydrophobic
pocket. And third, according to the structural studies of other
The model of the bound substrate reveals that the carboxylate and the
amide oxygen atoms occupy the positions of acetate oxygen atoms as
expected. The amide nitrogen forms a hydrogen bond with Ser289 O (2.8 Å), and the amide carbon is in close
proximity to the predicted water molecule. The side chain packs into
the hydrophobic pocket surrounded by Thr290,
Phe191, Lys252, Met254, and
Met347, in which Leu298, Tyr344,
and Met346 constitute the pocket base. The substrate
methionine side chain C Ligand-mediated Conformational Switch--
The
D-aminoacylase structure here seems a closed conformation,
because the active-site cavity is almost inaccessible to solvent (Fig.
5). The zinc ions lie in the deepest part
of the active site, and the hydrophobic side chain is close to the
opening of the pocket. The narrow opening of the cavity is capped by
the 63-residue insertion. The 63-residue insertion borders the active site and contains many putative substrate-interacting residues as
mentioned above. This domain may act as a gate controlling access to
the active site, affecting both substrate access and product release.
Particularly, the two antiparallel
This type of conformational switch upon the substrate binding is also
observed in other The Proposed Catalytic Mechanism--
The strong structural
homology of the
The crystal structure of D-aminoacylase with the modeled
substrate provides the structural basis for the enzyme catalytic mechanism. Together with the similar mechanisms in the Conclusion--
In summary, the crystal structure of
D-aminoacylase reveals that the enzyme indeed belongs to
the -barrel, and a catalytic (
)8-barrel with a
63-residue insertion. The enzyme structure shares significant similarity to the
/
-barrel amidohydrolase superfamily, in
which the
-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One
zinc ion is coordinated by Cys96, His220, and
His250, while the other is loosely chelated by
His67, His69, and Cys96. This is
the first example of the metal ion coordination by a cysteine residue
in the superfamily. Therefore, D-aminoacylase defines a
novel subset and is a mononuclear zinc metalloenzyme but containing a
binuclear active site. The preferred substrate was modeled into a
hydrophobic pocket, revealing the substrate specificity and enzyme
catalysis. The 63-residue insertion containing substrate-interacting
residues may act as a gate controlling access to the active site,
revealing that the substrate binding would induce a closed conformation
to sequester the catalysis from solvent.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
-barrel amidohydrolase superfamily (10,
11). And the metal ligands in D-aminoacylases have been proposed based on structural prediction (10) and mutational studies
(10, 12).
)8-barrel with conserved metal ligands,
four histidines and one aspartate, at the C-terminal ends of strands
1 (HXH),
5 (His),
6 (His), and
8
(Asp), and led to discovery of the
/
-barrel amidohydrolase
superfamily. The five metal ligands are strictly conserved and define a
subtle but sharp sequence signature in this superfamily.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Statistics of data collection and structural refinement
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-barrel
(residues 7-61 and 425-480) and a catalytic
/
-barrel (residues
62-414) (Fig. 1). There are two
insertions in the
/
-barrel: residues 147-165 between the
3
strand and
3-helix, forming a helix and a random coil, and residues
285-347 between
7 and
7, forming two helices and four
strands. The latter 63-residue insertion across the active site is
involved in the substrate-mediated conformational change (see below).
There are two large loops between
2 and
2 (residues 99-127) and
between
8 and
8 (residues 366-398).
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Fig. 1.
The A. faecalis
D-aminoacylase structure. A,
ribbon diagram of the C backbone. The structural fold contains a
small
-barrel comprising both the N and the C terminus (residues
7-61 and 425-480), a central
/
-barrel (residues 62-414), with
a 63-residue insertion (residues 285-347). The small
-barrel may be
responsible for the structural stabilization, the
/
-barrel for
catalysis, and the insertion for substrate-mediated conformational
switch. The tightly bound zinc ion, the metal ligands, and two acetate
molecules are shown as a pink sphere and
ball-and-stick representation. B, a close-up view
from the top of the elliptically distorted
/
-barrel. The tightly
bound zinc ion is chelated by Cys96, His220,
His250, and the first acetate.
/
-barrel amidohydrolase superfamily mentioned above (13-19), in particular, urease and cytosine deaminase, with r.m.s.1 deviations of
2.9 Å (325 C
atoms with 18% sequence identity) and 3.8 Å (330 C
atoms with 12% sequence identity), respectively. Structural
comparison reveals that the
-strands in both the small
barrel
and the catalytic
/
-barrel correspond closely, whereas the
external helices and surface-exposed loops diverge significantly. Despite the apparent lack of sequence similarity, the eight
-strands (56 structurally equivalent residues) of the
/
-barrels overlay within 1.2-2.0 Å r.m.s. deviations (Fig.
2A). On the other hand, the
size of the small
-domain, comprising both the N and the C termini,
varies greatly in different structures. The
-strands of the
-domains (48 structurally equivalent residues) overlay within
1.0-1.3 Å r.m.s. deviations (Fig. 2B). The small
-barrel does not contribute any residues to the active site and
appears to play a structural role.
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Fig. 2.
Structural superposition of
D-aminoacylase (green), urease
(red, Protein Data Bank code 1UBP), dihydroorotase
(pink, Protein Data Bank code 1J79),
dihydropyrimidinase (blue, Protein Data Bank code
1GKP), cytosine deaminase (yellow, Protein Data Bank
code 1K6W), adenosine deaminase (orange, Protein Data
Bank code 1A4M), and phosphotriesterase homology protein
(black, Protein Data Bank code 1BF6), with a close
view of the elliptically distorted
/
-barrel (A) and the
small
-barrel (B). The two
nickel ions in urease at the
and
sites are shown as red
spheres. The letters N and C indicate the N
and C termini in D-aminoacylase. The
-strands in both
barrels superimpose quite well.
(2.24 Å),
His220 N
1 (2.08 Å), His250
N
2 (2.04 Å), and an acetate molecule, ACT1
O2 (2.04 Å), from the crystallization solution.
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Fig. 3.
The metal center. A,
the 2Fo Fc electron density
map in the zinc center contoured at the 3
level and is shown in
green, and the weak density in the 2Fo
Fc map for the loosely bound zinc ion contoured at
the 2
level and is shown in purple. The structural
refinement revealed that the enzyme binds two zinc ions with very
different affinities. B, superposition of the bi-nickel
center in urease (Protein Data Bank code 1UBP), the mononuclear iron
center in cytosine deaminase (Protein Data Bank code 1K6W), and the
bi-zinc center in D-aminoacylase, shown in red,
blue, and green, respectively. The
metal-binding site is more buried, while the
site is more
solvent-exposed. The residue numbering is labeled in the same color for
each protein. The critical hallmark for the binuclear subset is a
carboxylated lysine residue serving as a bridging ligand. A cysteine
residue (Cys96) in D-aminoacylse, and the third
conserved histidine (His214) in cytosine deaminase,
compensate the missing carboxylated lysine.
/
-barrel amidohydrolase superfamily (13-19). A small
electron density at this site is observed after structural refinement
(Fig. 3A). The electron density at this site becomes much
stronger in the crystals soaking with 100 mM zinc acetate.
In addition, the zinc content of the purified recombinant DA1
D-aminoacylase was measured to be between 1.3-1.5 g·atom
per mole of enzyme (10). Therefore, the weak electron density is
assigned as zinc, which is tetrahedrally ligated by His67
Ne2 (2.01 Å), His69 N
2 (2.05 Å), Cys96 S
(2.19 Å), and ACT1
O1 (2.26 Å), and separated from the tightly bound zinc by
3.1 Å. The crystallographic refinement resulted in an occupancy of
0.25 with B-factor of 19.3 Å2 for the loosely
bound zinc ion and an occupancy of 1.0 with B-factor of 7.3 Å2 for the tightly bound zinc.
-lactamase from Bacillus cereus (26-28).
-lactamases share significant sequence identity (34%)
with highly conserved metal ligands, the enzyme from Bacteroides fragilis has a binuclear zinc center with similar metal affinities (Kd ~10 µM), whereas the B. cereus enzyme binds zinc ions with very distinct affinities
(Kd ~1 µM and 25 mM, respectively). The crystal structures suggested that the weak metal
binding may be due to the local electrostatic environment (26), and the
B. cereus enzyme functionally behaves as a monozinc enzyme
and may be an evolutionary intermediate between the mono- and bi-zinc
metallo-
-lactamases (27, 28).
/
-Barrel Amidohydrolase
Superfamily--
To date, there are two subsets in the
/
-barrel
amidohydrolase superfamily based on the metal centers with four
conserved histidines and one aspartate (Fig. 3B). In the
binuclear subset (13-17), the more buried metal ion (
site) is
coordinated by the first two conserved histidines from the common
zinc-binding HXH motif (29), the conserved aspartate,
and two bridging ligands, whereas the more solvent-exposed metal ion
(
site) is chelated by the other two conserved histidines and the
bridging ligands, consisting of a carboxylated lysine (or a glutamate)
and one water molecule (or a hydroxide ion). On the other hand, in the
second subset (18, 19), the metal is bound only at the
site ligated by the first three conserved histidines and one water molecule.
site, and the other binds weakly at the
site. This is the first example of a cysteine residue (Cys96) that coordinates to a
zinc ion in this superfamily (Fig. 3B). Mutational and
atomic absorption spectroscopic studies revealed that this cysteine
residue contributes the most toward the interactions with the zinc ions
among the ligands, because the mutant C96A shows the least zinc binding
affinity (10). Therefore, the unique metal center of
D-aminoacylase defines a novel subset, in which two metal
ions bind to the binuclear metal center with different affinities and
are bridged by a thiolate ligand (cysteine) instead of a carboxylate
ligand (carboxylated lysine or glutamate).
4 strand in the
/
-barrel (15; Fig. 2A). The
zinc-zinc distance of 3.1 Å in D-aminoacylase is
similar to the nickel-nickel distance of 3.1 Å in the
-mercaptoethanol (
-ME)-inhibited Bacillus
pasteurii urease (30), but it is significantly shorter than those
(3.4-3.8 Å) in the other crystal structures of the binuclear members
(13-17, 31). A screening of the Cambridge Structural Data base reveals
that the bridging thiolate sulfur atoms would shorten the di-metal
distance. Therefore, the shorter di-metal distance in
D-aminoacylase and in
-ME urease can be considered as an
intrinsic property of the metallic core.
(2.7 Å), Arg377 N
1 (2.7 Å),
N
2 (3.0 Å), Tyr283 O
(2.8 Å), and Ser289 N (2.8 Å), suggesting that this acetate
molecule occupies the binding position of the substrate carboxylate
group. Around this region, there is a hydrophobic pocket formed by the
side chains of Phe191, Tyr192,
Lys252, Met254, Leu298,
Tyr344, Met346, and Met347.
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Fig. 4.
The putative substrate-binding site.
A, the two acetate-binding sites in the active site
cavity (Act1 and Act2). Residues surrounded the
binding site are displayed as ball-and-stick representations
and the zinc ion as a purple sphere. B, the
proposed substrate-binding site with the modeled
N-acetyl-D-methionine in
ball-and-stick and the active water molecule
(Wat) as a green sphere. The oxygen atoms of the
acetates and substrate are expected to bind at the same position
because of the extensive interactions. The first acetate may displace
the attacking water molecule at the metal center.
/
-barrel amidohydrolase superfamily members (13-19, 31), the
amide oxygen atom coordinates the
ion (which is likely at the ACT1
O2 position), and the amide carbon atom must be in close
proximity to the active water molecule (the ACT1 O1
position). After modeling, the energy minimization was performed by
CNS (21) as the structural refinement.
and S
have close contacts with
Leu298 C
1 (3.3 Å), C
2 (3.2 Å), Tyr344 C
2 (3.4 Å), Thr290
C
2 (3.4 Å), and Phe191 C
(4.2 Å). In particular, Leu298, directly facing toward the
substrate, may be important for the substrate specificity, because the
D-aminoacylases with glutamate or aspartate preference
contain an arginine residue at this position.
-strands (residues 287-293 and
339-346) may act as the fulcrum of the conformational change, because
substrate-contacting residues are located in these regions. The closed
conformation described here may be due to the interaction between the
second acetate ACT2 and Tyr283 and Ser289,
sealing the entrance. Then the substrate binding would induce a closed
conformation to sequester the reaction complex from solvent.
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Fig. 5.
The closed conformation of the
D-aminoacylase. A, the protein
surface colored by electrostatic potential from 40 kBT
(red) to 40 kBT (blue) and
shown without the insertion. The entrance to the active site is open as
the 63-residue insertion is removed. The substrate
N-acetyl-D-methionine and the tightly bound zinc
ion are also shown as a stick model and a
purple sphere in the active site pocket,
respectively. B, the active site is closed with the
presence of the insertion. The protein is shown as ribbons
with the small
-domain, the
/
-barrel, and the insertion
colored blue, cyan, and green,
respectively. The surfaces of the
/
-barrel and the insertion are
shown only for regions near the active site. The view of B
is rotated 45 degrees about the vertical axis.
/
-barrel amidohydolases, including adenosine
deaminase (18), cytosine deaminase (19), and urease (30, 31). In these
three enzymes, the conformational changes appear to be induced by
direct contacts between protein and the bound substrate. In adenosine
deaminase and cytosine deaminase, similar flaps are formed by the
insertions between the
1-strand and the
1-helix. On the other
hand, in urease, the lid is formed from the insertion between
7 and
7, as that in the D-aminoacylase. It is worth noting
that the enzyme inhibition mechanism of D-aminoacylase by
acetate may be similar to that of urease by
-ME, because in both
cases, one inhibitor molecule bridges the metal ions and another
molecule induces a closed conformation. In the
-ME-inhibited urease,
one
-ME ligates the two nickel ions, and another
-ME forms a
mixed disulfide with Cys322 sealing the entrance (30).
/
-barrel amidohydrolases is also reflected in
their catalytic mechanisms, in particular, preparation of the active
nucleophile for the hydrolytic reaction is very similar (13-19, 31).
The
metal ion functions in activation of the nucleophile water by
lowering its pKa, while the
metal ion serves as
an electrophilic catalyst to polarize the carbonyl-oxygen bond of the
substrate. The highly conserved Asp366 probably acts as a
general base to activate the nucleophile water. The proximity of
His67 N
1 to Asp366
O
1 (3.2 Å) could facilitate the proton abstraction and
donation, and the proximity of His69 N
2 to
ACT1 O1 (3.1 Å), and to Asp366
O
1 (3.3 Å), might further assist in activating the
attacking water molecule and stabilizing the negatively charged intermediate.
/
-barrel amidohydrolases, we propose a catalytic mechanism for
D-aminoacylase in Scheme 1.
First, Asp366 abstracts the proton from the water molecule,
and the tightly bound zinc ion polarizes the carbonyl-oxygen bond, thus
facilitating the nucleophilic attack on the amide carbon atom to form
the tetrahedral intermediate. Cleavage of the carbon-nitrogen bond is
assisted by the simultaneous protonation of the amide nitrogen. The
newly formed acetate then ligates the zinc ion. An
N-acetyl-L-methionine substrate can also be
modeled into the active site; however, for the L-isomer the
interaction between the substrate amide and Ser289 backbone
carbonyl would be missing, perhaps resulting in lack of proper
orientation of the amide carbon for water attacking, then with thus 100 times lower hydrolysis efficiency than the D-form substrate
(2).
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Scheme 1.
The
proposed catalytic mechanism for
D-aminoacylase.
/
-barrel amidohydrolase superfamily and defines a novel
subset. A putative substrate-binding pocket with key residues is
identified. The unusual 63-residue large insertion involves in the
substrate specific recognition and the active-site entrance switch. On
the basis of our structural information, some protein engineering
trials such as deletion of the small
-domain and change of the
substrate specificity by using mutagenesis are under investigation.
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ACKNOWLEDGEMENTS |
---|
The synchrotron radiation experiments were performed at the Synchrotron Radiation Research Center, Hsinchu, Taiwan, at the Photon Factory, Tsukuba, Japan, and at the SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (Proposal No. 2002A0504-CL1-np).
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FOOTNOTES |
---|
* This work was supported by National Science Council Grants NSC 91-2311-B-010-010, NSC 90-2321-B-002-002, and NSC 90-2321-B-001-015.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1M7J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§§ To whom correspondence may be addressed: Inst. of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan. E-mail: ahjwang@gate.sinica.edu.tw (for A. H.-J. W.) or Inst. of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan. Tel.: 886-2-2826-7278; Fax: 886-2-2820-2449; E-mail: shliaw@ym.edu.tw (for) Y.-C. T.).
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M210795200
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ABBREVIATIONS |
---|
The abbreviations used are:
r.m.s., root mean
square;
-ME,
-mercaptoethanol;
kBT, Boltzmann's constant × temperature (1.38 × 10
23 J/K × T = 1.38 × 10
23 J).
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REFERENCES |
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