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.
Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile
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Josep-Lluís VILADOT, Francesc CANALS, Xavier BATLLORI, Antoni PLANAS; Long-lived glycosyl-enzyme intermediate mimic produced by formate re-activation of a mutant endoglucanase lacking its catalytic nucleophile. Biochem J 1 April 2001; 355 (1): 79–86. doi: https://doi.org/10.1042/bj3550079
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