NMR spectroscopy was used to search for mechanistically significant differences between the thermodynamic and dynamic properties of the 34 kDa (alpha/beta)(8)-barrel catalytic domain of beta-(1,4)-glycosidase Cex (or CfXyn10A) in its free (apo-CexCD) and trapped glycosyl-enzyme intermediate (2FCb-CexCD) states. The main chain chemical shift perturbations due to the covalent modification of CexCD with the mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside are limited to residues within its active site. Thus, consistent with previous crystallographic studies, formation of the glycosyl-enzyme intermediate leads to only localized structural changes. Furthermore, N-15 relaxation methods demonstrated that the backbone amide and tryptophan side chains of apo-CexCD are very well ordered on both the nanosecond to picosecond and millisecond to microsecond time scales and that these dynamic features also do not change significantly upon formation of the trapped intermediate. However, covalent modification of CexCD led to the increased protection of many amides and indoles, clustered around the active site of the enzyme, against fluctuations leading to hydrogen exchange. Similarly, thermal denaturation studies demonstrated that 2FCb-CexCD has a significantly higher midpoint unfolding temperature than apo-CexCD. The covalently modified protein also exhibited markedly increased resistance to proteolytic degradation by thermolysin relative to apo-CexCD. Thus, the local and global stability of CexCD increase along its reaction pathway upon formation of the glycosyl-enzyme intermediate, while its structure and fast time scale dynamics remain relatively unperturbed. This may reflect thermodynamically favorable interactions with the relatively rigid active site of Cex necessary to bind, distort, and subsequently hydrolyze glycoside substrates.