Refined Molecular Structure of Pig Pancreatic α-Amylase at 2·1 Å Resolution

Abstract

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 eight 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 hydrophobic 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 dextrins 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 enzymelogical 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.