1RP8: Crystal Structure Of Barley Alpha-Amylase Isozyme 1 (Amy1) Inactive Mutant D180a In Complex With Maltoheptaose

Citation:
Abstract
Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."
PDB ID: 1RP8Download
MMDB ID: 33521
PDB Deposition Date: 2003/12/3
Updated in MMDB: 2007/10
Experimental Method:
x-ray diffraction
Resolution: 2  Å
Source Organism:
Similar Structures:
Biological Unit for 1RP8: monomeric; determined by author
Molecular Components in 1RP8
Label Count Molecule
Protein (1 molecule)
1
Alpha-amylase Type 1 Isozyme(Gene symbol: LOC548204)
Molecule annotation
Chemicals (20 molecules)
1
16
2
1
3
3
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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