Calcium binding in alpha-amylases: an X-ray diffraction study at 2.1-A resolution of two enzymes from Aspergillus.
X-ray diffraction analysis (at 2.1-A resolution) of an acid alpha-amylase from Aspergillus niger allowed a detailed description of the stereochemistry of the calcium-binding sites. The primary site (which is essential in maintaining proper folding around the active site) contains a tightly bound Ca2+ with an unusually high number of eight ligands (O delta 1 and O delta 2 of Asp175, O delta of Asn121, main-chain carbonyl oxygens of Glu162 and Glu210, and three water molecules). A secondary binding site was identified at the bottom of the substrate binding cleft; it involves the residues presumed to play a catalytic role (Asp206 and Glu230). This explains the inhibitory effect of calcium observed at higher concentrations. Neutral Aspergillus oryzae (TAKA) alpha-amylase was also refined in a new crystal at 2.1-A resolution. The structure of this homologous (over 80%) enzyme and additional kinetic studies support all the structural conclusions regarding both calcium-binding sites.
Chemistry Department, University of York, Heslington, U.K.
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E
Boel
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Brady
A M
AM
Brzozowski
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Derewenda
G G
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Dodson
V J
VJ
Jensen
S B
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Petersen
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Swift
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Thim
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Woldike
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004247:DNA; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D014961:X-Ray Diffraction; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D004247:DNA; D000516:alpha-Amylases; D002118:Calcium