26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis.
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.
Ohnishi
Tsuneo
T
Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara, 631-8505.
Juffer
André H
AH
Tamoi
Masahiro
M
Skriver
Karen
K
Fukamizo
Tamo
T
D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D018506:Gene Expression Regulation, Plant; D001467:Hordeum / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D007477:Ions; D002688:Chitinases