Journal of Biochemistry Advance Access originally published online on July 15, 2009
Journal of Biochemistry 2009 146(5):651-657; doi:10.1093/jb/mvp110
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Importance of the Hydrogen Bonding Network Including Asp52 for Catalysis, as Revealed by Asn59 Mutant Hen Egg-white Lysozymes
1Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, 812-8582; 2Aino University, 4-5-4 Higashi-ohta, Ibaragi, Osaka 567-0012; and 3Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-77, Japan
*To whom correspondence should be addressed. Tel/Fax: +81-92-642-6998, E-mail: kmaenaka-umin{at}umin.net
Correspondence may also be addressed to Dr Izumi Kumagai. Tel: +81-22-795-7274, Fax: +81-22-795-6164, E-mail: kmiz{at}mail.tains.tohoku.ac.jp
Received April 27, 2009; Accepted June 29, 2009
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Abstract |
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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.
Key Words: catalytic activity, lysozyme, protein–carbohydrate interactions, protein engineering, X-ray crystallography
Abbreviations: PNP, p-nitrophenyl; GlcNAc, N-acetyl-β-D-glucosaminine (PNP-G1cNAc); MurNAc, N-acetylmuramic acid; SDS, sodium dodecyl sulfate; HPLC, high pressure liquid chromatography; NMR, nuclear magnetic resonance