Molecular Modelling of the Interaction Between the Catalytic Site of Pig Pancreatic α‐Amylase and Amylose Fragments

Abstract

A stereo chemical refinement of the crystalline complex between porcine pancreatic a-amylase and a pseudopentasaccharide from the amylostatin family has been performed through molecular mechanics calculations, using a set of parameters appropriate for protein and protein-carbohydrate interactions. The refinement provided a starting point for docking a maltopentaose moiety within the catalytic site, in the absence of water. A thorough exploration of the different orientations and conformations of maltopentaose established the sense of binding of the amylosic substrate in the amylase cleft. After optimising the geometry of the binding site, the conformations adopted by the four contiguous linkages could be rationalised by considering the environment, either hydrophobic or hydrophilic, of the different glucose moieties. Seemingly, details of the non-bonded interactions (hydrogen bonds, van der Waals and stacking interactions) that underlie this molecular recognition have been established. In particular, it was confirmed that the three acidic amino acids of the catalytic site (Asp197, Asp300 and Glu233) are close to their glucosidic target, and that there is no steric reason to propose an alteration of the 4C, conformation of the glucose residue prior to hydrolysis. However, in the absence of water molecules, it is difficult to elucidate the details of the catalysis. Additional macroscopic information has been gained, such as the impossibility to fit a double-helical arrangement of amylose chains in the amylasic cleft. This explains why some native starches containing such motifs resist amylolytic enzymes. Tentative models involving longer amylosic chains have been elaborated, which extend our knowledge of the interaction and orientation of starch fragments in the vicinity of the hydrolytic sites.

Automated docking of monosaccharide substrates and analogues and methyl α-Acarviosinide in the glucoamylase active site

“…The problem has been addressed by limited spatial searching coupled with energy minimization to study the interaction between proteins and monosaccharides 23,24 and between enzymes and flexible carbohydrate chains. [25][26][27] Systematic spatial searches of the binding modes of monosaccharides to concavalin A have been performed using extended atom representation for hydroxyl groups. 28 Many different approaches used to search the spatial and conformational space available to ligands in their interaction with proteins were reviewed in recent docking studies.…”

Amylose chain behavior in an interacting context II. Molecular modeling of a maltopentaose fragment in the barley ?-amylase catalytic site

“…This substrate conformation is now compared to another pentasaccharide fragment modeled in the catalytic site of PPA. 52 However, one must beware of the differences in ring labeling when referring to equivalent subsite numbering. In PPA, the rings are labeled from A PPA to E PPA but the corresponding subsites span from (؊3) to (؉2).…”

“…The superimposition of these structures only on the basis of the backbone atoms of these catalytic residues reveals 3D topographical homologies for some other conserved residues. In terms of stacking phenomena, Y51 (subsite ؊1) is tightly bound to ring A as does equivalent Y PPA 62 with the C PPA ring 52 or could do Y TAA 82 with the corresponding glucose TAA ring. Similarly, H92 and H288, 55 which are involved in hydrogen bonds with ring A, are known to play an important role in the endo-type specificity as do the same amino acid residues in PPA (H PPA 101 and H PPA 299) or in TAA (H TAA 122 and H TAA 296).…”

Specificity Modulation of Barley α-Amylase through Biased Random Mutagenesis Involving a Conserved Tripeptide in β → α Loop 7 of the Catalytic (β/α)8-Barrel Domain

“…Insight into the mechanism and substrate interactions at the long binding site was gained from structures of complexes of (i) substrates with inactive mutants of R-amylase (34,48), cyclodextrin glycosyltransferase (39,49,50), and maltotetraohydrolase (51), (ii) active enzymes and inhibitory maltooligosaccharide analogues (20,21,26,28,35,49,50,52), and (iii) substrate docking in R-amylases (24,53). Throughout the binding crevice sugar rings stacking onto aromatic side chains and hydroxyl groups are forming hydrogen bonds with the protein and with structural water molecules.…”