Insights into transition state stabilization of the beta-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants.
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Notenboom V, Birsan C, Nitz M, Rose DR, Warren RA, Withers SG
Insights into transition state stabilization of the beta-1,4-glycosidase Cex by covalent intermediate accumulation in active site mutants.
Nat Struct Biol. 1998 Sep;5(9):812-8.
- PubMed ID
- 9731776 [ View in PubMed]
- Abstract
The catalytic mechanism of 'retaining' beta-glycosidases has been the subject of considerable interest and debate for many years. The visualization of a covalent glycosyl enzyme intermediate by X-ray crystallography was first accomplished with a saccharide substrate substituted with fluorine at its 2-position. The structure implicated major roles for residue His 205 and for the 2-hydroxyl position of the proximal saccharide in binding and catalysis. Here we have studied the kinetic behavior of various His 205 mutants. One of these mutants, a double mutant H205N/E127A, has been used to stabilize a covalent glycosyl-enzyme intermediate involving an unsubstituted sugar, permitting crystallographic analysis of the interactions between its 2-hydroxyl group and the enzyme.