Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile.

The mutant E134A 1,3-1,4-beta-glucanase from Bacillus licheniformis, in which the catalytic nucleophilic residue has been removed by mutation to alanine, has its hydrolytic activity rescued by exogenous formate in a concentration-dependent manner. A long-lived alpha-glycosyl formate is detected and identified by (1)H-NMR and matrix-assisted laser desorption ionization-time-of-flight-MS. The intermediate is kinetically competent, since it is, at least partially, enzymically hydrolysed, and able to act as a glycosyl donor in transglycosylation reactions. This transient compound represents a true covalent glycosyl-enzyme intermediate mimic of the proposed covalent intermediate in the reaction mechanism of retaining glycosidases.