STRUCTURAL IMPLICATION OF TWO SACCHARIDE CONFORMATIONS IN THE ACTIVE SITE OF A ß-GLYCOSIDASE A. White^*, D. Tull^[[section]], K. L. Johns^*, S. G. Withers^[[section]], and D. R. Rose^*; *Department of Medical Biophysics, University of Toronto and Ontario Cancer Institute, Toronto, Canada M5G 2M9; [[section]]Department of Chemistry, University of British Columbia, Vancouver, Canada V6T 1Z1.

It is well established from biochemical and structural studies that a saccharide deformation takes place during catalysis by glycosyl hydrolases. In the context of two catalytic carboxylates in the active site of most of the retaining ß-1,4-glycosidases, the hydrolysis is believed to proceed by a double displacement catalytic mechanism through a covalent intermediate with oxocarbenium transition states. Our previous crystallographic studies revealed that a covalent a-glycosyl-enzyme catalytic intermediate can be accommodated in the confined space of the active site of the enzyme cex-cd. [White, A., et al., 1996, Nature Struct. Biol. 3:149] In this structure the attached saccharide adopts a chair conformation which differs from the planar arrangement of the transition states. We report here the structure of cex-cd complexed with the inhibitor cellobial (1,2-ene-1,2-dideoxy-cellobiose) designed to mimic the sugar conformation of the transition states.

Soaking of a crystal of the bacterial xylanase/cellulase cex-cd in 20 mM cellobial induces less than 0.2% change to the P4₁2₁2 unit cell parameters. Data to 2.2 Å resolution were collected using a SDMW area detector and then reduced to an R-merge of 0.07 . The crystal structure of the unliganded cex-cd [White, A., et al., 1994, Biochemistry 33:12546] was used to solve its complexed form. At the current stage of refinement with X-PLOR the R-factor is 0.175 with a free R value of 0.26, given a data to parameters ratio of 1.4 and good model stereochemistry. The difference Fourier electron density map reveals a prominent element of electron density in the active site of cex-cd, indicating the presence of a cellobial molecule. Compared to the fluorocellobiosyl covalent complex [White et al., 1996], the cellobial occupies the same subsites and makes a similar network of interactions between the distal saccharide and the enzyme. Further comparison of the structure of these liganded forms of cex-cd may inform on the catalytic mechanism of retaining ß-glycosidases.

Supported by the Protein Engineering Network of Centres of Excellence.