26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis
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26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. / Ohnishi, Tsuneo; Juffer, André H; Tamoi, Masahiro; Skriver, Karen; Fukamizo, Tamo.
I: Journal of Biochemistry, Bind 138, Nr. 5, 2005, s. 553-62.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - 26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis
AU - Ohnishi, Tsuneo
AU - Juffer, André H
AU - Tamoi, Masahiro
AU - Skriver, Karen
AU - Fukamizo, Tamo
N1 - Keywords: Amino Acid Sequence; Catalysis; Catalytic Domain; Chitinase; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Plant; Hordeum; Hydrogen-Ion Concentration; Ions; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Structure-Activity Relationship
PY - 2005
Y1 - 2005
N2 - To explore the structure essential for the catalysis in 26 kDa endochitinase from barley seeds, we calculated theoretical pKa values of the ionizable groups based on the crystal structure, and then the roles of ionizable side chains located near the catalytic residue were examined by site-directed mutagenesis. The pKa value calculated for Arg215, which is located at the bottom of the catalytic cleft, is abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. No enzymatic activity was found in the Arg215-mutated chitinase (R215A) produced by the Escherichia coli expression system. The transition temperature of thermal unfolding (T(m)) of R215A was lower than that of the wild type protein by about 6.2 degrees C. In the crystal structure, the Arg215 side chain is in close proximity to the Glu203 side chain, whose theoretical pKa value was found to be abnormally low (-2.4), suggesting that these side chains may interact with each other. Mutation of Glu203 to alanine (E203A) completely eliminated the enzymatic activity and impaired the thermal stability (deltaT(m) = 6.4 degrees C) of the enzyme. Substrate binding ability was also affected by the Glu203 mutation. These data clearly demonstrate that the Arg215 side chain interacts with the Glu203 side chain to stabilize the conformation of the catalytic cleft. A similar interaction network was previously found in chitosanase from Streptomyces sp. N174 [Fukamizo et al. (2000) J. Biol. Chem. 275, 25633-25640]; hence, this type of interaction seems to be at least partly conserved in the catalytic cleft of other glycosyl hydrolases.
AB - To explore the structure essential for the catalysis in 26 kDa endochitinase from barley seeds, we calculated theoretical pKa values of the ionizable groups based on the crystal structure, and then the roles of ionizable side chains located near the catalytic residue were examined by site-directed mutagenesis. The pKa value calculated for Arg215, which is located at the bottom of the catalytic cleft, is abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. No enzymatic activity was found in the Arg215-mutated chitinase (R215A) produced by the Escherichia coli expression system. The transition temperature of thermal unfolding (T(m)) of R215A was lower than that of the wild type protein by about 6.2 degrees C. In the crystal structure, the Arg215 side chain is in close proximity to the Glu203 side chain, whose theoretical pKa value was found to be abnormally low (-2.4), suggesting that these side chains may interact with each other. Mutation of Glu203 to alanine (E203A) completely eliminated the enzymatic activity and impaired the thermal stability (deltaT(m) = 6.4 degrees C) of the enzyme. Substrate binding ability was also affected by the Glu203 mutation. These data clearly demonstrate that the Arg215 side chain interacts with the Glu203 side chain to stabilize the conformation of the catalytic cleft. A similar interaction network was previously found in chitosanase from Streptomyces sp. N174 [Fukamizo et al. (2000) J. Biol. Chem. 275, 25633-25640]; hence, this type of interaction seems to be at least partly conserved in the catalytic cleft of other glycosyl hydrolases.
U2 - 10.1093/jb/mvi154
DO - 10.1093/jb/mvi154
M3 - Journal article
C2 - 16272567
VL - 138
SP - 553
EP - 562
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 5
ER -
ID: 8878454