Secondary tritium and primary 14C kinetic isotope effects were measured for the hydrolysis of α-D-glucopyranosyl fluoride catalyzed by sugar beet seed α-D-glucosidase, forming α-D-glucose, and by Rhizopus niveus glucoamylase forming β-D-glucose. The data provided a novel opportunity to model and directly compare the transition state structures for the hydrolysis of a substrate promoted with retention or inversion of configuration according to the enzyme catalyst. The isotope effects for the reaction catalyzed by each enzyme are most consistent with an S(N)1 rather than an S(N)2 mechanism. The modeled transition state structures for the hydrolysis promoted by the α-glucosidase and the glucoamylase both bear significant oxocarbonium ion character, with the D-glucosyl residue having a flattened 4C1 conformation and a C-1-O-5 bond order of 1.92, even though opposite D- glucose anomers were produced from the substrate. The transition states show some modest differences, but their general similarity strongly suggests that the stereochemical outcome of glycosylase reactions does not predict the transition state structure, nor does the transition state structure of such reactions predict the stereochemical outcome. The results support previously reported evidence for the separate topological control of product configuration by protein structures in these and other glycosylases.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology