CRYSTALLOGRAPHIC COMPLEXES OF GLUCOAMYLASE WITH MALTOOLIGOSACCHARIDE ANALOGS - RELATIONSHIP OF STEREOCHEMICAL DISTORTIONS AT THE NONREDUCING END TO THE CATALYTIC MECHANISM

Crystal structures at pH 4 of complexes of glucoamylase from Aspergill us awamori var. X100 with the pseudotetrasaccharides D-gluco-dihydroac arbose and acarbose have been refined to R-factors of 0.147 and 0.131 against data to 1.7 and 2.0-Angstrom resolution, respectively, The two inhibitors bind in nearly identical manners, each exhibiting a dual b inding mode with respect to the location of the last wgar residues. Th e reduced affinity of D-gluco-dihydroacarbose (K-i = 10(-8) M) relativ e to acarbose (K-i = 10(-12) M) may stem in part from the weakening of hydrogen bonds of the catalytic water (Wat 500) to the enzyme. Steric contacts between the nonreducing end of D-gluco-dihydroacarbose and t he catalytic water perturb Wat 500 from its site of optimal hydrogen b onding to the active site. Interactions within the active site displac e the 6-hydroxymethyl group of the nonreducing end of both acarbose an d D-gluco-dihydroacarbose toward a more axial position. In the case of D-gluco-dihydroacarbose the shift in the position of the 6-hydroxymet hyl group occurs with a 12 degrees change in two dihedral angles of th e glucopyranose ring toward a half-chair conformation The observed con formational distortion of the first residue of D-gluco-dihydroacarbose is consistent with the generation of a glucopyranosyl cation in the t ransition state. Comparable distortions of stereochemistry in model co mpounds require approximately 2 kcal/mol, not more than 25% of the ene rgy necessary to form the half-chair conformation in glucose, The magn itude of stereochemical distortion observed in the active site of gluc oamylase suggests that favorable electrostatic interactions between th e putative glucopyranosyl cation intermediate and the active site must be more important in stabilizing the transition state than mechanical distortion of the substrate.