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 wh ich the catalytic nucleophilic residue has been removed by mutation to alan ine, has its hydrolytic activity rescued by exogenous formate in a concentr ation-dependent manner. A long-lived alpha -glycosyl formate is detected an d identified by H-1-NMR and matrix-assisted laser desorption ionization-tim e-of-flight-MS. The intermediate is kinetically competent, since it is, at least partially. enzymically hydrolysed, and able to act as a glycosyl dono r in transglycosylation reactions. This transient compound represents a tru e covalent glycosyl-enzyme intermediate mimic of the proposed covalent inte rmediate in the reaction mechanism of retaining glycosidases.