| Autor: |
Larson, SB, Greenwood, A, Cascio, D, Day, J, McPherson, A |
| Jazyk: |
angličtina |
| Rok vydání: |
1994 |
| Zdroj: |
Larson, SB; Greenwood, A; Cascio, D; Day, J; & McPherson, A. (1994). Refined molecular structure of pig pancreatic α-amylase at 2.1 Å resolution. Journal of Molecular Biology, 235(5), 1560-1584. doi: 10.1006/jmbi.1994.1107. UC Irvine: Retrieved from: http://www.escholarship.org/uc/item/59j9f1pz |
| DOI: |
10.1006/jmbi.1994.1107. |
| Popis: |
The structure of pig pancreatic α-amylase has been determined by X-ray diffraction analysis using multiple isomorphous replacement in a crystal of space group P212121 (a = 70.6 Å, b = 114.8 Å, c = 118.8 Å) containing nearly 75% solvent. The structure was refined by simulated annealing and Powell minimization, as monitored by 2Fo-Fc difference Fourier syntheses, to a conventional R of 0.168 at 2.1 Å resolution. The final model consists of all 496 amino acid residues, a chloride and a calcium ion, 145 water molecules and an endogenous disaccharide molecule that contiguously links protein molecules related by the 21 crystallographic operator along x. The protein is composed of a large domain (amino acid residues 1 to 403) featuring a central α/β-barrel of bight parallel strands and connecting helices with a prominent excursion between strand β3 and helix α3 (amino acid residues 100 to 168). The final 93 amino acid residues at the carboxyl terminus form a second small domain consisting of a compact Greek key β-barrel. The domains are tightly associated through hydrophohic interfaces. The β3/α3 excursion and portions of the central α/β-barrel provide four protein ligands to the tightly bound Ca ion; three water molecules complete the coordination. The Cl- ion is bound within one end of the α/β-barrel by two arginine residues in a manner suggesting a plausible mechanism for its allosteric activation of the enzyme. A crystalline complex of the pancreatic α-amylase with α-cyclodextrin, a cyclic substrate analog of six glucose residues, reveals, in difference Fourier maps, three unique binding sites. One of the α-cyclodextrin sites is near the center of the long polysaccharide binding cleft that traverses one end of the α/β-barrel, another is at the extreme of this cleft. By symmetry this can also be considered as two half sites located at the extremes of the active site cleft. This latter α-cyclodextrin displaces the endogenous disaccharide when it binds and, along with the first sugar ring, delineates the extended starch binding site. The third α-cyclodextrin binds at an "accessory site" near the edge of the protein and is quite distant from the polysaccharide binding cleft. Its presence explains the multivalency of α-amylase binding to dextrine in solution. The extended active site cleft is formed by large, sweeping, connecting loops at one end of the α/β-barrel. These include three sequence segments that are highly conserved among α-amylases. Residues suggested by kinetic and enzymological studies to participate in catalysis are found in the depths of the binding cleft close to the α-cyclodextrin bound near its center. Superposition of the lysozyme active site cleft upon that of the α-amylase produces virtual coincidence of the lysozyme catalytic residues, Glu35 and Asp52, upon Glu233 and Asp300 of α-amylase. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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