Mutational analysis of amino acid residues involved in catalytic activity of a family 18 chitinase from tulip bulbs

Article abstract of DOI:10.1271/bbb.67.341

We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.

Full text of DOI:10.1271/bbb.67.341

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Mutational Analysis of Amino Acid Residues Involved
in Catalytic Activity of a Family 18 Chitinase from
Tulip Bulbs
Keisuke SUZUKAWAa, Takeshi YAMAGAMIa, Takayuki OHNUMAa, Hideki HIRAKAWAb, Satoru
KUHARAb, Yoichi ASOa & Masatsune ISHIGUROa
a Laboratories of Protein Chemistry and Engineering, Kyushu University6-10-1 Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan
b Genetic Regulation, Graduate School of Bioresource and Bioenvironmental Sciences,
Kyushu University6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
Published online: 22 May 2014.
To cite this article: Keisuke SUZUKAWA, Takeshi YAMAGAMI, Takayuki OHNUMA, Hideki HIRAKAWA, Satoru KUHARA, Yoichi
ASO & Masatsune ISHIGURO (2003) Mutational Analysis of Amino Acid Residues Involved in Catalytic Activity of a Family 18
Chitinase from Tulip Bulbs, Bioscience, Biotechnology, and Biochemistry, 67:2, 341-346, DOI: 10.1271/bbb.67.341
To link to this article: http://dx.doi.org/10.1271/bbb.67.341
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Biosci. Biotechnol. Biochem., 67 (2), 341–346, 2003
Mutational Analysis of Amino Acid Residues Involved in Catalytic Activity
of a Family 18 Chitinase from Tulip Bulbs
2
Keisuke SUZUKAWA,1 Takeshi YAMAGAMI,1 Takayuki OHNUMA,1 Hideki HIRAKAWA
,
†
1,
Satoru KUHARA,2 Yoichi ASO,1 and Masatsune ISHIGURO
2
1Laboratories of Protein Chemistry and Engineering and Genetic Regulation, Graduate School
of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
Received August 16, 2002; Accepted October 6, 2002
chitinases have three conserved disulˆde bonds.9)
We expressed chitinase-1 (TBC-1) from tulip bulbs
Tulipa bakeri ) in E. coli cells and used site-directed
(
Bulb chitinases do not lyse Micrococcus luteus cell
walls but hevamine10) and PLC-B11) do lyse such cell
walls. Hydrolysis of (GlcNAc)5 yields mainly
(GlcNAc)3 and (GlcNAc)2 by bulb chitinases, but
PLC-B hydrolyzes (GlcNAc)5 mainly to (GlcNAc)4
and GlcNAc. We propose therefore that bulb
chitinases should be classiˆed into a new subclass,
mutagenesis to identify amino acid residues essential for
catalytic activity. Mutations at Glu-125 and Trp-251
completely abolished enzyme activity, and activity
decreased with mutations at Asp-123 and Trp-172 when
glycolchitin was the substrate. Activity changed with the
mutations of Trp-251 to one of several amino acids with
side-chains of little hydrophobicity, suggesting that
hydrophobic interaction of Trp-251 is important for the
activity. Molecular dynamics (MD) simulation analysis
with hevamine as the model compound showed that the
distance between Asp-123 and Glu-125 was extended by
mutation of Trp-251. Kinetic studies of Trp-251-mutat-
ed chitinases conˆrmed these various phenomena. The
results suggested that Glu-125 and Trp-251 are essential
for enzyme activity and that Trp-251 had a direct role in
ligand binding.
class IIIb, within class III.5,6) Elsewhere, we cloned
12)
TBC-1 cDNA and overexpressed it in E. coli
.
To
understand the structural basis for activity of class
IIIb chitinase, enzymatic characterization, molecular
dynamics simulation, and kinetic analysis of mutated
TBCs-1 prepared by site-directed mutagenesis at six
amino acid residues were done.
Materials and Methods
Materials. TBC-1 was prepared from tulip (Tulipa
bakeri) bulbs as described previously.6) GlcNAc
oligomers were prepared by the method of Rupley.13)
Oligonucleotides were purchased from Amersham
Pharmacia Biotech. Restriction endonucleases and
DNA-modifying enzymes were from Takara Shuzo
or New England Biolabs. The plasmid vectors used
were as follows: pBluescript II from Stratagene,
pGEM-T Easy Vector from Promega, and pET-22b
expression vector from Novagen. A Quikchange
site-directed mutagenesis kit was purchased from
Stratagene. A ‰uorescence-labeled primer cycle se-
quencing kit (Thermo Sequenasetm) with 7-deaza-
dGTP was from Amersham. All other chemicals
were of analytical grade for biochemical use.
Key words: chitinase; tulip bulb; Tulipa bakeri; site-
directed mutagenesis
Plant chitinases (EC 3.2.1.14) catalyze the hydro-
lysis of b-1,4-linked homopolymers or oligomers of
GlcNAc; the enzymes seem to be self-defense-related
proteins for protection against fungal pathogens.1–3)
Class III chitinase has no sequence similarity to class
I or II chitinases but is similar in sequence to bacteri-
al and fungal chitinases.4) We previously isolated two
bulb chitinases, GBC-a from gladiolus (Gladiolus
5)
gandavensis
)
and TBC-1 from tulip (Tulipa
bakeri ),6) and characterized their enzymatic proper-
ties and amino acid sequences,7,8) ˆnding that bulb
chitinases are in class III. There are, however, diŠer-
ences in properties between bulb chitinases and plant
class III chitinases characterized so far. Bulb
chitinases have no disulˆde bonds, but plant class III
Enzymatic activity and kinetics. The assay of
chitinase activity and calculation of steady-state
kinetic parameters were done colorimetrically with
†
To whom correspondence should be addressed. Beppu University, College of Food and Nutrition, 82 Kita-Ishigaki, Beppu 874-8501,
Japan; Fax: +81-977-66-9631; E-mail: ishiguro mc.beppu-u.ac.jp
Abbreviations: TBC-1, chitinase-1 of tulip bulbs; GBC-a, chitinase-a from gladiolus bulbs; PLC-B, pokeweed leaf chitinase-B; rTBC-1,
recombinant TBC-1; NBS, -bromosuccinimide; MD, molecular dynamics
＠
N

342
K. SUZUKAWA et al.
Table 1. Primers Used to Construct Recombinant DNA and TBC-1 Mutants
a
Primer
S9A-5
Sequence (5?ª3?)
?
?
5
?
-CGTGAGTACATCGGCGCGCAGTTTAACGATGTC-3
-CCGTGAGTACATCGGCCAGCAGTTTAACGATGTCAAG-3
-GTACCATCTTGATGGAATTGAAATCGACTACGAGCAC-3
-CAGTACCATCTTGATGGAATTAATATCGACTACGAGCAC-3
-GATGGAATTGATATCGAATACGAGCACTTCAAAGGTGAC-3
-GATGGAATTGATATCAACTACGAGCACTTCAAAGGTGAC-3
-GAATTGATATCGACTACCAGCACTTCAAAGGTGACC-3
-GGAATTGATATCGACTACGATCACTTCAAAGGTGACC-3
-GCCACTACCAGGCGTTGGCGGAGAAGTACG-3
-GGGGAAGCTTCATGGCATCTTTGTTTTCTCGGCTGATG-3
-CATCAGCCGAGTAAACAAAGATGCCATGAAGCTTCCCC-3
-GGAAGCTTCATGGCATCTTTGTTTTGTCGGCTGATG-3
-GCATCTTTGTTGCGTCGTCGGCTGATG-3
?
S9Q-5
D121E-5
D121N-5
D123E-5
D123N-5
E125Q-5
E125D-5
W172A-5
W251F-5
W251Y-5
W251L-5
W251A-5
5
?
?
?
?
?
?
?
?
?
?
?
5
?
5
?
?
?
?
5
?
5
?
5
?
?
5
?
?
5
?
?
5
?
?
?
?
?
5
?
?
5
?
?
5?
?
a
Underlined codons encode amino acid changes.
glycolchitin as the substrate. The enzymatic reaction
was monitored in terms of changes in the amount
of reducing sugars by the method of Imoto and
Yagishita.14) Ten microliters of the enzyme solution
MD simulation. MD simulation of hevamine and
three mutants, W255F, W255L, and W255Y, was
done with the Discover 95.0 program (Accerlrys
Inc.). The structure of each mutant was modeled on
the basis of the X-ray crystal structure of hevamine
(PDB code, 1HVQ). The parameter used was the
consistent valence Forceˆeld (cvŠ). The parameters
are provided for amino acids, water, and a variety of
other functional groups. After Trp-255 of hevamine
was replaced with the InsightII package (Accerlrys
Inc.), the optimum dihedral angles of mutated side
chains were searched for by avoidance of steric con-
‰icts. Next, the following three steps were applied for
energy minimization. First, the coordinates of the
heavy atoms of proteins were tethered loosely
(1,047 kJmol„1 nm„1) and the positions of hydrogen
atoms were optimized by the steepest-descents
method and conjugate gradients method until the
(1.6 pmol) was added to 500
chitin solution in 0.1 sodium acetate buŠer, pH
5.0. After incubation at 37 C for 15 min, the reduc-
ml of 0.2z (w v) glycol-
W
M
9
tion by the mixture was measured with ferri-ferroc-
yanide reagent. One unit of activity was deˆned as
the enzyme activity that produced 1
mmol of GlcNAc
per minute at 37 C. Concentrations of TBC-1,
9
recombinant TBC-1 (rTBC-1), and mutant proteins
were measured by amino acid analysis.
Site-directed mutagenesis. Mutants of TBC-1 were
generated by site-directed mutagenesis of the ap-
propriate regions of the wild-type chitinase gene,
tbc-1, with the mutagenesis kit. The nomenclature of
TBC-1 mutants and the oligonucleotide primers used
for mutagenesis are shown in Table 1. Mutations
were introduced into an ampliˆed mature TBC-1
cDNA fragment that had been subcloned into the
pGEM-T Easy Vector. After mutagenesis, the ma-
ture fragment was sequenced to verify the presence of
maximum derivative was less than 2.1 kJmol„1 nm„1
Second, the coordinates of the main-chain atoms of
proteins were tethered loosely (1,047 kJmol„1 nm„1
.
)
and the positions of the amino acid residues were
optimized. Third, the positions of all atoms were
optimized without being tethered until the maximum
the desired mutation, excised by digestion with Nde
I
derivative was less than 2.1 kJmol„1 nm„1
.
and BamHI, and ligated into an expression vector,
pET-22b, that had been digested with the same en-
zymes. The mutant plasmids were used to transform
E. coli BL21 (DE3). All mutant forms and rTBC-1
were obtained from culture media in soluble form
and were puriˆed to homogeneity by the method as
reported previously.12)
Next, the structure was ˆlled by overlaying a
12.0-Å layer of single point charge (SPC) water with
the SOAK procedure of the InsightII program. Be-
fore MD simulation was started, the following two
steps were applied for energy minimization. First, all
atoms of proteins were ˆxed and the positions of
hydrogen atoms of water molecules were optimized
by the steepest-descents method until the maximum
derivative was less than 2.1 kJmol„1 nm„1. Second,
the all atoms of proteins were ˆxed while the posi-
tions of water molecules were optimized until the
maximum derivative of water molecules was less than
CD spectra of native TBC-1 and its mutant pro-
teins. Far ultraviolet CD spectra of the chitinases
were obtained with
a
JASCO J-720 spec-
tropolarimeter. The spectra were measured at protein
concentrations of 10 in 50 m sodium phosphate
buŠer, pH 7.0, with a 0.1-cm cell path length at
25 C.
m
M
M
2.1 kJmol„1 nm„1
.
The conformation optimized by the energy
minimization was used as a starting conformation for
NVT (N, number of particles, V, constant volume
9

Site-directed Mutagenesis of Tulip Bulb Chitinase-1
343
Fig. 2. SDS-PAGE Analysis of rTBC-1 and Its Mutant Proteins.
Homogeneity of the rTBC-1 and its mutant proteins was con-
ˆrmed by SDS-PAGE as described by Laemli. Lanes 1-15 con-
tained rTBC-1, S9A, S9Q, D121N, D121E, D123N, D123E,
E125Q, E125D, W172A, rTBC-1, W251F, W251Y, W251L, and
W251A, respectively, and M contained bovine serum albumin
(66 kDa), ovalbumin (45 kDa), wild-type TBC-1 (31 kDa), and
myoglobin (17.2 kDa).
Fig. 1. Mono-Q Column Chromatography (pH 7.5) of Chitinase
Fractions Obtained in Sephacryl S-200HR Gel Filtration.
The chitinase fractions obtained in Sephacryl S-200HR gel
chromatography of chitinase mutant preparations expressed in
E. coli were separately put on a Mono-Q column equilibrated
(not shown). All mutant proteins were overexpressed
to the same extent as rTBC-1, the yields were close to
25 mg of protein from the soluble fraction in 1 l of
the culture.
with 10 m
M
Tris-HCl buŠer (pH 7.5). Elution was done with a
in the same buŠer.
linear gradient of NaCl from zero to 0.3
M
The solid line indicates the elution proˆle of rTBC-1 (280 nm),
and arrows indicate the positions of the peaks where each
mutant protein was eluted. The names of the mutant proteins
are given in Table 1.
Characterization of mutant proteins
The speciˆc activities toward glycolchitin of the
and T, constant temperature) molecular dynamics at
300 K to generate possible stable conformations.
The system was warmed to 300 K. After a 100-fs
equilibration stage, simulation was continued at
300 K for 300 ps with a 1.0-fs time step. The simula-
tions were done with the Verlet velocity algorithm15)
and the structures were stored in the computer every
1.0 ps.
enzymes were 106
94.5 for S9Q, 32.7
D121E, 8.5 for D123N, 1.0
E125Q, and E125D, 72.5 for W172A, 89.8
W251F, 50.0 for W251Y, 2.2
for W251A, when the enzyme activity of the
native TBC-1 was taken to be 100 . The Glu-125 of
z
for rTBC-1, 100
for D121N, 64.8
for D123E, 0
z
for S9A,
z
z
z
for
for
for
z
z
z
z
z
z
z for W251L, and
0
z
z
TBC-1, which corresponds to the catalytic residue
Glu-127 in hevamine (only the three-dimensional
structure of which has been solved as a class III
chitinase), was changed to glutamine (E125Q). The
Trp-251 of TBC-1, which corresponds to Trp-255 of
hevamine being related to substrate binding, and
which was rapidly oxidized with NBS at pH 4.0 in
TBC-1,16) was replaced with phenylalanine, tyrosine,
leucine, and alanine, respectively. The proteins
E125Q, E125D, W251A, and W251L had no
chitinase activity. When Trp-251 was replaced with
phenylalanine, tyrosine, leucine, and alanine, the
chitinase activity decreased in proportion to the
decrease in hydrophobicity of the side chain of the
amino acid residues introduced. When Asp-123,
which corresponds another catalytic carboxylate in
hevamine, was changed to asparagine or glutamic
acid, activity decreased much, but a change at
Asp-121, which might be a catalytic carboxylate
positioned near the catalytic center, to asparagine or
glutamic acid caused a moderate loss of activity. A
moderate eŠect was observed when Trp-172, which
was one of the ˆrst rapidly reacting tryptophan
residues with NBS, was changed to alanine. No activ-
ity loss was observed when Ser-9 was replaced with
alanine or glutamine.
Results
Construction and expression of TBC-1 mutants,
and puriˆcation of mutant proteins
To identify the amino acid residues responsible for
the chitinase activity of TBC-1, 13 TBC-1 mutants
were engineered by site-directed mutagenesis and
expressed in E. coli cells. The mutated amino acid
residues were selected by comparison of the amino
acid sequences of TBC and hevamine as well as the
result of NBS-oxidation16) of TBC-1. The resulting
mutant proteins were independently puriˆed from
the soluble fraction of the sonic extracts by the proce-
dures described for rTBC-1.12) The elution proˆles
are shown in Fig. 1 as mutant E125Q, D123N, and
D121N were eluted earlier than rTBC-1, which was
eluted at the same position as wild TBC-1, but the
mutant proteins (E125D, D121E and D123E) were
eluted later from the Mono-Q column. Other mutant
proteins were eluted at the same position as rTBC-1.
Figure 2 shows SDS-PAGE proˆles of a typical
preparation of rTBC-1 and its mutant puriˆed pro-
teins. All mutant proteins gave CD spectra similar to
the spectrum of TBC-1, suggesting that the expressed
proteins were unaltered from the wild-type enzyme

344
K. SUZUKAWA et al.
Table 2. Average Intermolecular Distances between Asp-125 and
Glu-127 in the Active Sites of Wild-type and Three Mutant Pro-
teins
Ca„Ca: Asp-125Ca„Glu-127Ca (Å)
Wild-type
6.40
W255F
6.67
W255Y
6.48
W255L
6.70
Mean
Hydrogen bond: Asp-125OH„Glu-127O2 (Å)
Wild-type
2.04
W255F
2.95
W255Y
3.81
W255L
5.59
Mean
The intermolecular distances, Asp-125Ca-Glu-127Ca and Asp-125OH-
Glu-127O2, were calculated using the sampled structures by MD simulation.
Table 3. Kinetic Parameters of Wild-type and Tryptophan-mu-
tated TBC-1
Fig. 3. Backbone Structures of Hevamine and Its Mutant Forms
Seen by MD Simulation.
Enzyme
Km (mg ml)
kcat (s„1
)
kcat Km (ml s mg)
WW
The main chain was drawn with a blue line for wild-type heva-
mine, a yellow line for W255F, a green line for W255Y, and a
violet line for W255L. The structures of the mutant forms have
been superimposed on that of the wild-type enzyme, and amino
acid residues responsible for the active site of hevamine are
shown in orange.
W
W
Wild-type
W251F
W251Y
W251L
0.72
0.86
1.56
2.93
0.76
0.54
0.36
0.02
1.06
0.63
0.23
0.01
MD simulation
sists of Glu-127 and Asp-125 for catalytic function
and Trp-255 for ligand binding.17)
Asp-123, Glu-125, and Trp-251 of TBC-1 cor-
responded to Asp-125, Glu-127, and Trp-255 of
hevamine in sequence alignment, respectively. The
main-chain of the wild type and the main chains of
the mutant proteins, W255F, W255Y, and W255L
were quite similar (Fig. 3). On the other hand, the
distance between Glu-127 and Asp-125, which may
be in the catalytic site of chitinase, seemed to increase
when Trp-255 was replaced with phenylalanine,
tyrosine, or leucine. The distance calculated between
Ser-9 in TBC-1 is an alanine in TBC-2 with a
speciˆc activity 1 50 that of TBC-1, and Ser-9 cor-
W
responds to a glutamine in hevamine, which residue
seems to be hydrogen-bonded to a substrate, so Ser-9
was mutated here to alanine or glutamine. Because
Asp-121, Asp-123, and Glu-125 were in conserved
positions in class III chitinases, these residues
were mutated to asparagine (D121N), glutamic
acid (D121E), asparagine (D123N), glutamic acid
(D123E), glutamine (E125Q) and aspartic acid
(E125D). As the oxidation of Trp-172 and Trp-251
with NBS resulted in the complete loss of enzymatic
activity,16) and as the importance of the Trp residues
in other plant chitinases was reported,20) Trp-172 and
Trp-251 were mutated. CD spectral determination of
all mutant proteins suggested that the overall struc-
tures were not altered (not shown). The relative
enzyme activities of the mutant forms showed that
Asp-123 and Glu-125 were responsible for catalytic
activity and that Trp-251 was needed for substrate
binding. The chitinase activity of the Trp-251 mutant
enzymes decreased in proportion to the decrease in
hydrophobicity of the side chains of the amino acid
residues that changed. Mutation of Ser-9 to alanine
or glutamine had little eŠect on chitinase activity,
suggesting that this residue was not essential; Ser-9
may not be involved in hydrogen bonding as it is in
hevamine. The eŠects of a mutation of Asp-121
could be related to disturbance of the hydrophobic
ˆeld around the corresponding Asp-123 of hevamine
by Tyr-6. Because this ˆeld was not formed around
one of the active site residue, chitinase activity was
a
-carbons of two acidic amino acids (Asp-125 and
Glu-127) showed that the main chain of hevamine
changed little with a mutation of Trp-255 with
phenylalanine, tyrosine, or leucine (Table 2). In fact,
however, the distance between side chains of Asp-125
and Glu-127 was increased by mutation.
Kinetic parameters
The replacement of Trp-251 (Trp-255 of hevamine)
with phenylalanine, tyrosine, or leucine increased Km
and decreased kcat, respectively (Table 3).
Discussion
Of plant class III chitinases, hevamine from Hevea
brasiliensis has been studied in most detail, including
X-ray crystallographic analysis of hevamine-sub-
strate ((GlcNAc)3) complexes17) and hevamine-inhibi-
tor (allosamidin) complexes.18) Hevamine has been
classiˆed as a family 18 glycosyl hydrolase, with a (
b
a
)8 barrel structure with an active center that cata-
W
lyzes hydrolysis of
b
-1,4-glycosidic bonds by a sub-
strate-assisted mechanism.19) This active center con-

Site-directed Mutagenesis of Tulip Bulb Chitinase-1
345
by X-ray crystallographic analysis of (GlcNAc)
oligomer-TBC-1 complex are being in progress and
will in the future give more detail information on the
binding site of substrate to the active site of TBC-1
and on a local structure assisting the enzyme catalysis
from behind the catalytic center.
Acknowledgment
Computation time was provided by the Supercom-
puter Laboratory, Institute for Chemical Research,
Kyoto University. This study was partially supported
from the Research for the Future Program of the
Japan Society for the Promotion of Science.
Fig. 4. EŠects of Hydrophobicity of Side Chains at Residue 251
on Chitinase Activity of Mutant Enzymes.
References
The hydrophobicity (
D gt, cal mol) of amino acid residues
W
1) Schlumbaum, A., Mauxh, F., Vogeli, U., and Boller,
T., Plant chitinases are potent inhibitors of fungal
growth. Nature, 324, 365–367 (1986).
2) Brochaert, W. F., Van Pariji, J., Allen, A. K., and
Peumans, W. J., Comparison of some molecular, en-
zymatic, and antifungal properties of chitinases from
thorn-apple, tobacco, and wheat. Physiol. Mol. Plant
Pathol., 33, 319–331 (1988).
3) Stintzi, A., Heitz, T., Prasad, V., Wiedermann-
Merdinoglu, S., KauŠmann, S., GeoŠroy, P.,
Legrand, M., and Fritig, B., Plant ``pathogenesis-
related'' proteins and their role in defense against
pathogens. Biochimie, 75, 687–706 (1993).
4) Henrissat, B., and Bairoch, A., New families in the
classiˆcation of glycosyl hydrolases based on amino
acid sequence similarities. Biochem. J., 293, 781–788
(1993).
introduced into TBC-1 (abscissa) was plotted against chitinase
activity (left ordinate) and the simulated distance between side
chains of Asp-123 and Glu-125 (right ordinate). The hydropho-
bicity of tryptophan, phenylalanine, tyrosine, and leucine are
taken from Nozaki and Tanford.21)
indirectly in‰uenced when Asp-121 was mutated to
asparagine.
No study on the role of the Trp-255 residue of
hevamine (Trp-251 of TBC-1) by site-directed muta-
genesis has appeared, although it has been reported
that Trp-255 is important for the activity of heva-
mine. MD simulation of TBC-1 mutant forms sug-
gested that the decrease of chitinase activity of
W251F, W251Y, and W251L corresponded to the
diŠerent hydrophobicity of their side chains,
although the main chains of the mutated forms
seemed unaltered. These results suggested that
Trp-251 of TBC-1 interacted with a substrate via
hydrophobic force. Another important result is that
the distance between the side chains of Asp-123 and
Glu-125 (Asp-125 and Glu-127 of hevamine), both
residues of which are needed for activity, was greater
when the side chains of amino acids introduced were
more hydrophobic (Fig. 4). These results showed that
Trp-251 indirectly governed the spatial positions of
Asp-123 and Glu-125. Correct positioning would
make possible interactions needed for catalysis. Ki-
netic parameters were compatible with our sugges-
5) Yamagami, T., Mine, Y., Aso, Y., and Ishiguro, M.,
Puriˆcation and characterization of two chitinase
isoforms from the bulbs of gladiolus (Gladiolus
gandavensis). Biosci. Biotechnol. Biochem.
2140–2142 (1997).
, 61,
6) Yamagami, T., Taira, T., Aso, Y., and Ishiguro, M.,
Isolation and characterization of chitinase isoforms
from the bulbs of four species of the genus Tulipa.
Biosci. Biotechnol. Biochem., 62, 584–587 (1998).
7) Yamagami, T., Mine, Y., and Ishiguro, M.,
Complete amino acid sequence of chitinase-a from
bulbs of gladiolus (Gladiolus gandavensis). Biosci.
Biotechnol. Biochem., 62, 386–389 (1998).
8) Yamagami, T., and Ishiguro, M., Complete amino
acid sequences of chitinase-1 and -2 from bulbs of ge-
tions above. The Km for W251L was fourfold that for
nus Tulipa. Biosci. Biotechnol. Biochem.
1253–1257 (1998).
9) Tanigawa, M., Yamagami, T., and Funatsu, G., The
complete amino acid sequence of chitinase-B from the
leaves of pokeweed (Phytolacca americana). Biosci.
Biotechnol. Biochem., 59, 841–847 (1995).
, 62,
the wild type, suggesting that a‹nity for binding
decreased; kcat decreased with mutation. Hydropho-
bic stacking, especially of aromatic rings, seemed
therefore to be needed for substrate binding. At the
same time, an amino acid residue with high
hydrophobicity was needed for correct distance and
orientation of Asp-123 and Glu-125 for catalytic ac-
tivity.
10) Jeckel, P. A., Hartmann, J. B. H., and Beintema, J.
J., The primary structure of hevamine, an enzyme
with lysozyme chitinase activity from Hevea
W
brasiliensis. Eur. J. Biochem., 200, 123–130 (1991).
11) Ohta, M., Yamagami, T., and Funatsu, G., Puriˆca-
tion and characterization of two chitinases from the
Experiments on hydrolysis of (GlcNAc)6 by TBC-1
and Trp-mutated TBC-1 and on the precise position-
ing of substrates in the active center of this enzyme

346
K. SUZUKAWA et al.
leaves of pokeweed (Phytolacca americana). Biosci.
Biotechnol. Biochem., 59, 656–661 (1995).
12) Yamagami, T., Tsutsumi, K., and Ishiguro, M.,
Cloning, sequencing, and expression of the tulip bulb
chitinase-1 cDNA. Biosci. Biotechnol. Biochem., 64,
1394–1401 (2000).
17) Terwisscha van Scheltinga, A. C., Kalk, K. H.,
Beintema, J. J., and Dijkstra, B. W., Crystal
structures of hevamine: a plant defense protein with
chitinase and lysozyme activity, and its complex with
an inhibitor. Structure, 2, 1181–1189 (1994).
18) Terwisscha van Scheltinga, A. C., Armand, S., Kalk,
K. H., Isogai, A., Henrissat, B., and Dijkstra, B. W.,
13) Rupley, J. A., The hydrolysis of chitin by concentrat-
ed hydrochloric acid, and the preparation of low-
molecular weight substrates for lysozyme. Biochim.
Biophys. Acta, 83, 245–255 (1964).
Stereochemistry of chitin hydrolysis by
a plant
chitinase lysozyme and X-ray structure of a complex
W
with allosamidin: evidence for substrate assisted
catalysis. Biochemistry, 34, 15619–15623 (1995).
19) Fukamizo, T., Chitinolytic enzymes: catalysis, sub-
strate binding, and their application. Curr. Proteins
Peptide Sci., 1, 105–124 (2000).
14) Imoto, T., and Yagishita, K., A simple activity meas-
urement of lysozyme. Agric. Biol. Chem.
1154–1156 (1971).
, 35,
15) Swope, W. C., Anderson, H. C., Berens, P. H., and
Wilson, K. R., A computer simulation method for the
calculation of equilibrium constants for the forma-
tion of physical clusters of molecules: application to
small water clusters. J. Chem. Phys., 76, 637–649
(1982).
16) Ishiguro, M., Yamagami, T., Tanigawa, M.,
Tsutsumi, K., Funatsu, G., Ohnuma, T., and Aso,
Y., Modiˆcation of tryptophan residues of plant class
III chitinases involved in enzymatic activity. J. Fac.
Agr. Kyushu Univ., 46, 243–250 (2001).
20) Yamagami, T., and Funatsu, G., Involvements of
Trp23 in the chitin-binding and of Trp131 in the
chitinase activity of rye seed chitinase-a. Biosci.
Biotechnol. Biochem., 61, 1819–1825 (1997).
21) Nozaki, Y., and Tanford, C., The solubility of amino
acids and two glycine peptides in aqueous ethanol and
dioxane solutions. J. Biol. Chem., 246, 2211–2217
(1971).