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.
Laboratori de Bioquímica, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.
J L
JL
Viladot
F
F
Canals
X
X
Batllori
A
A
Planas
D001407:Bacillus / Q000201:enzymology; D002240:Carbohydrate Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D005561:Formates / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005561:Formates; C030544:formic acid; D006026:Glycoside Hydrolases; C028020:licheninase