26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis.

26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. Research Abstract Details 

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26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. Abstract Text:

Tsuneo Ohnishi, Juffer,Masahiro Tamoi,Karen Skriver,Tamo Fukamizo,

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.

26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. Publishing Authors By Initials

T Ohnishi,AH Juffer,M Tamoi,K Skriver,T Fukamizo,

For similar biochemical phenomena, metabolism, and nutrition: biochemical phenomena: structure-activity relationship research abstracts see: biochemical phenomena, metabolism, and nutrition: biochemical phenomena: structure-activity relationship research

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26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 138

Page Numbers: 553-62

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Nov

YEAR: 2005

26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. Information

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LANGUAGE: eng

NlmUniqueID: 376600

26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. Keywords Mesh Terms:

KEYWORDS: Structure-Activity Relationship

MESH TERMS: genetics

Chemical & Substance for Abstract: 26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis. Information

Substance Name: Chitinase

Registry Number: EC 3.2.1.14

Grant and Affiliation Information for 26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis.

AFFILIATION: Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara, 631-8505.

Country: Japan

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MEDLINETA: J Biochem

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