TY - JOUR
T1 - Structure of the Aspergillus oryzae α-amylase complexed with the inhibitor acarbose at 2.0 Å resolution
AU - Brzozowski, A. Marek
AU - Davies, Gideon J.
PY - 1997/9/9
Y1 - 1997/9/9
N2 - The three-dimensional structure of the Aspergillus oryzae α-amylase (TAKA-amylase), in complex with the inhibitor acarbose, has been determined by X-ray crystallography at a resolution of 1.98 Å. The tetrasaccharide inhibitor is present as a hexasaccharide presumably resulting from a transglycosylation event. The hexasaccharide occupies the -3 to +3 subsites of the enzyme, consistent with the known number of subsites determined by kinetic studies, with the acarbose unit itself in the -1 to +3 subsites of the enzyme. The transition state mimicking unsaturated pseudo-saccharide occupies the -1 subsite as expected and is present in a distorted 2H3 half- chair conformation. Careful refinement plus extremely well-resolved unbiased electron density suggest that the hexasaccharide represents a genuine transglycosylation product, but the possibility that this apparent species results from an overlapping network of tetrasaccharides is also discussed. Catalysis by α-amylase involves the hydrolysis of the α-1,4 linkages in amylose with a net retention of the anomeric configuration, via a double- displacement mechanism, as originally outlined by Koshland [Koshland, D. E. (1953) Biol. Rev. 28, 416-336]. The enzymatic acid/base and nucleophile, residues Glu230 and Asp206, respectively, are appropriately positioned for catalysis in this complex, and the hexasaccharide species allows mapping of all the noncovalent interactions between protein and ligand through the enzyme's six subsites.
AB - The three-dimensional structure of the Aspergillus oryzae α-amylase (TAKA-amylase), in complex with the inhibitor acarbose, has been determined by X-ray crystallography at a resolution of 1.98 Å. The tetrasaccharide inhibitor is present as a hexasaccharide presumably resulting from a transglycosylation event. The hexasaccharide occupies the -3 to +3 subsites of the enzyme, consistent with the known number of subsites determined by kinetic studies, with the acarbose unit itself in the -1 to +3 subsites of the enzyme. The transition state mimicking unsaturated pseudo-saccharide occupies the -1 subsite as expected and is present in a distorted 2H3 half- chair conformation. Careful refinement plus extremely well-resolved unbiased electron density suggest that the hexasaccharide represents a genuine transglycosylation product, but the possibility that this apparent species results from an overlapping network of tetrasaccharides is also discussed. Catalysis by α-amylase involves the hydrolysis of the α-1,4 linkages in amylose with a net retention of the anomeric configuration, via a double- displacement mechanism, as originally outlined by Koshland [Koshland, D. E. (1953) Biol. Rev. 28, 416-336]. The enzymatic acid/base and nucleophile, residues Glu230 and Asp206, respectively, are appropriately positioned for catalysis in this complex, and the hexasaccharide species allows mapping of all the noncovalent interactions between protein and ligand through the enzyme's six subsites.
UR - http://www.scopus.com/inward/record.url?scp=0030760550&partnerID=8YFLogxK
U2 - 10.1021/bi970539i
DO - 10.1021/bi970539i
M3 - Article
C2 - 9283074
AN - SCOPUS:0030760550
SN - 0006-2960
VL - 36
SP - 10837
EP - 10845
JO - Biochemistry
JF - Biochemistry
IS - 36
ER -