Importance of the Hydrogen Bonding Network Including Asp52 for Catalysis, as Revealed by Asn59 Mutant Hen Egg-white Lysozymes

In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel β-sheet, designated as a 'platform', o...

Full description

Saved in:
Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2009-11, Vol.146 (5), p.651-657
Main Authors: Ose, Toyoyuki, Kuroki, Kimiko, Matsushima, Masaaki, Maenaka, Katsumi, Kumagai, Izumi
Format: Article
Language:English
Subjects:
Acetylglucosamine - metabolism
Animals
Aspartic Acid - metabolism
Bacteriolysis
Biocatalysis
catalytic activity
Catalytic Domain
Chickens
Crystallography, X-Ray
Hydrogen Bonding
Hydrolysis
lysozyme
Muramidase - chemistry
Muramidase - metabolism
Mutant Proteins - chemistry
Mutant Proteins - metabolism
Mycobacterium - metabolism
protein engineering
Protein Structure, Secondary
protein–carbohydrate interactions
Structure-Activity Relationship
X-ray crystallography
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel β-sheet, designated as a 'platform', on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)₅. A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)₃ was bound in the A-B-C subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these 'platform' residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis.
ISSN:0021-924X
1756-2651
DOI:10.1093/jb/mvp110