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Structural analysis of mutant hen egg-white lysozyme preferring a minor binding mode
K Maenaka1, M Matsushima, G Kawai
1Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
Biochimica Et Biophysica Acta
|May 29, 1998
Summary
The Trp62His mutant hen lysozyme exhibits altered substrate binding, hydrolyzing inhibitors instead of binding them. This functional change stems from subtle protein-carbohydrate interaction shifts, not catalytic system alterations.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Tryptophan 62 (Trp62) in hen egg-white lysozyme is crucial for protein-carbohydrate interactions, involving stacking and hydrogen bonding.
- Previous studies indicated that substituting Trp62 alters substrate binding, with the Trp62His mutant showing a preference for a minor binding mode.
Purpose of the Study:
- To elucidate the functional and structural changes in the Trp62His mutant hen lysozyme.
- To analyze the complex structure of the Trp62His mutant with a substrate analogue using X-ray crystallography.
Main Methods:
- X-ray crystallography was employed to analyze the structure of the Trp62His mutant hen lysozyme complexed with N-acetylglucosamine trimer ((GlcNAc)3).
- Hydrolytic activity assays using synthetic substrates were performed to characterize the mutant's enzymatic properties.
Main Results:
- The overall protein structure of the Trp62His mutant was nearly identical to the wild-type, with His62 occupying a similar plane to wild-type Trp62.
- The Trp62His mutant hydrolyzed (GlcNAc)3, an inhibitor for the wild-type, and bound a product, N-acetylglucosamine dimer ((GlcNAc)2), in an unusual mode.
- The mutant enzyme was identified as a beta-anomer retaining enzyme, indicating conversion of the alpha-anomer product from a beta-anomer precursor.
Conclusions:
- The Trp62His mutation significantly alters substrate binding modes without changing the enzyme's catalytic system.
- Subtle rearrangements in the protein-carbohydrate interactions involving His62 and sugar residues B and C likely drive the observed functional changes.