X-ray Crystallographic Analyses of Pig Pancreatic α-Amylase with Limit Dextrin, Oligosaccharide, and α-Cyclodextrin^,

Abstract

Further refinement of the model using maximum likelihood procedures and re-evaluation of the native electron density map has shown that crystals of pig pancreatic α-amylase, whose structure we reported more than fifteen years ago, in fact contain a substantial amount of carbohydrate. The carbohydrate fragments are the products of glycogen digestion carried out as an essential step of the protein's purification procedure. In particular, the substrate-binding cleft contains a limit dextrin of six glucose residues, one of which contains both α-(1,4) and α-(1,6) linkages to contiguous residues. The disaccharide in the original model, shared between two amylase molecules in the crystal lattice, but also occupying a portion of the substrate binding cleft, is now seen to be a tetrasaccharide. There are, in addition, several other probable monosaccharide binding sites. To these results we have further reviewed our X-ray diffraction analysis of α-amylase complexed with α-cyclodextrin. α-Amylase binds three cyclodextrin molecules. Glucose residues of two of the rings superimpose upon the limit dextrin and the tetrasaccharide. The limit dextrin superimposes in large part upon linear oligosaccharide inhibitors visualized by other investigators. By comprehensive integration of these complexes we have constructed a model for the binding of polysaccharides having the helical character known to be present in natural substrates such as starch and glycogen. Keywordsα-amylase; hydrolase; α-cyclodextrin; X-ray crystallography; limit dextrin Pancreatic α-amylase (PPA, α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) was among the first enzymes to be extensively analyzed in terms of substrate binding and catalytic properties (1, 2) and one of the first enzymes to be crystallized (3), yet many of its most salient features remain a mystery. This is due largely to the range of substrates that it will degrade and the diverse structures that those substrates assume. α-Amylase hydrolyzes, without anomeric inversion, α-(1,4) linkages between glucose residues in (1) amylopectin and amylose, which comprise starch, (2) glycogen, (3) oligosaccharides of various lengths, and (4) some cyclodextrins. Both amylopectin and glycogen contain α-(1,6) linkages, which α-amylase † This work was supported by NIH grant GM 080412 to AM. ‡ Atomic coordinates and structure factors for the structures presented here have been deposited in the Protein Data Bank at the Research Collaboratory for Structural Bioinformatics with PDB codes 3L2L and 3L2M.

Diosgenin from Dioscorea bulbifera: Novel Hit for Treatment of Type II Diabetes Mellitus with Inhibitory Activity against α-Amylase and α-Glucosidase

“…The salient feature of α-amylase is presence of three highly conserved and catalytic residues such as Asp197, Glu233 and Asp300 in the active site pocket [54]. Similar types of hydrogen bonding and hydrophobic interactions have also been discussed in earlier crystal structures of α-amylase with different inhibitor molecules [55]–[57]. In accordance with fluorescence and CD studies the docking interaction showed involvement of tryptophan residue in catalytic pocket of α-amylase as reported in crystallography data [55]–[57].…”

Crystal Structures of Escherichia coli Branching Enzyme in Complex with Linear Oligosaccharides

“…This expectation is borne out by every GH13 enzyme/oligosaccharide structure known, all of which show the non-reducing end of the glucan emanating in this direction. (27,(46)(47)(48)(49) To illustrate this point, figure 1b shows an overlay of the composite EcBE structure with the oligomers from pullulanase (PDB 2FHF) bound in the active site modeled in EcBE.…”

Starch Hydrolase Inhibitors from Edible Plants

“…Both enzymes catalyze hydrolysis reaction via conjugated acid-based catalysis, but they have distinct substrate-binding sites. a-Amylase has a long grove filled with amino acid residues that can form multiple hydrogen bonds with oligosaccharides (Larson, Day, & McPherson, 2010), whereas the substratebinding sites for a-glucosidase are much smaller. The structural difference of the two enzymes enables selectivity of the inhibitors.…”