The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex

Acarbose is a naturally occurring pseudo-tetrasaccharide. It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases. To probe the interactions of ac arbose with other carbohydrate recognition enzymes, the crystal structure o f E. coli maltodextrin phosphorylase (MalP) complexed with acarbose has bee n determined at 2.95 Angstrom resolution and refined to crystallographic R- values of R (R-free) = 0.241 (0.293), respectively. Acarbose adopts a confo rmation that is close to its major minimum free energy conformation in the MalP-acarbose structure. The acarviosine moiety of acarbose occupies sub-si tes +1 and +2 and the disaccharide sub-sites +3 and +4. (The site of phosph orolysis is between sub-sites -1 and +1.) This is the first identification of subsites +3 and +4 of MalP. Interactions of the glucosyl residues in sub -sites +2 and +3 are dominated by carbohydrate stacking interactions with t yrosine residues. These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase. A glycerol molecule from the cryoprotectant occupies sub- site -1. The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the e xterior surface of MalP, provides a structural rationalization of the subst rate selectivity of MalP for a pentasaccharide substrate. Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranf erases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations. This flexibility allows acarbose to tar get a number of different enzymes. The two alternative conformations of aca rbose when bound to different carbohydrate enzymes are discussed.