Download MendeleyDirect Experimental Observation of the Hydrogen-Bonding Network of a Glycosidase Along its Reaction Coordinate Revealed by Atomic Resolution Analyses of Endoglucanase Cel5A
Varrot, A., Davies, G.J.(2003) Acta Crystallogr D Biol Crystallogr 59: 447
- PubMed: 12595701
- DOI: https://doi.org/10.1107/s0907444902023405
- Primary Citation of Related Structures:
1H11, 1H2J, 1HF6 - PubMed Abstract:
Non-covalent interactions between protein and ligand at the active centre of glycosidases play an enormous role in catalysis. Dissection of these hydrogen-bonding networks is not merely important for an understanding of enzymatic catalysis, but is also increasingly relevant for the design of transition-state mimics, whose tautomeric state, hydrogen-bonding interactions and protonation contribute to tight binding. Here, atomic resolution ( approximately 1 A) analysis of a series of complexes of the 34 kDa catalytic core domain of the Bacillus agaradhaerens endoglucanase Cel5A is presented. Cel5A is a 'retaining' endoglucanase which performs catalysis via the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. Previous medium-resolution analyses of a series of enzymatic snapshots has revealed conformational changes in the substrate along the reaction coordinate (Davies et al., 1998). Here, atomic resolution analyses of the series of complexes along the pathway are presented, including the 'Michaelis' complex of the unhydrolysed substrate, the covalent glycosyl-enzyme intermediate and the complex with the reaction product, cellotriose. These structures reveal intimate details of the protein-ligand interactions, including most of the carbohydrate-associated H atoms and the tautomeric state of crucial active-centre groups in the pH 5 orthorhombic crystal form and serve to illustrate the potential for atomic resolution analyses to inform strategies for enzyme inhibition.
Organizational Affiliation:
Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5YW, England.
