The mutant E134A 1,3-1,4-β-glucanase from Bacilluslicheniformis, 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 α-glycosyl formate is detected and identified by 1H-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.

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