Structural basis of alpha-amylase activation by chloride

Abstract

To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.

Fig. 1.

Schematic representation of the chloride binding site and of the interaction network with active site residues (adapted from Qian et al. 1994). The essential catalytic residues are in italics. Chloride ligands are Lys300(337), Arg172(195), Asn262(298), and H₂O1003(525); active site residues are Glu200(233), Asp174(197), and Asp264(300) the putative catalytic water molecule H₂O1004(cat) and His263(299). Numbers in parentheses refer to pig pancreas α-amylase.

Fig. 2.

Superposition of the chloride binding site in P. haloplanktis α-amylase (AHA), in green, complex AHA/nitrate in blue, mutant K300Q in yellow, and mutant K300R in pink.

Fig. 3.

Nitrate replacing chloride in the complex P. haloplanktis α-amylase (AHA)/NO₃⁻. Stereo view of the 2F_o − F_c electron density (in blue) after the insertion of the NO₃⁻ ion, showing its interactions with Arg172, Asn262, and Lys300.

Fig. 4.

Chloride binding site in the inactive mutant K300Q. 2F_o − F_c density, negative F_o − F_c density, and positive F_o − F_c density are shown in blue, green, and pink, respectively. Here phases have been generated before the replacement of Lys300 by Gln. It is clearly seen that the obtained structure is devoid of chloride by the positive F_o − F_c density and that the glutamine residue that replaces lysine at position 300 points away from the former binding site. Please notice that the glutamine replaces a water molecule (Wat1001) in the water pocket earlier described (Aghajari et al. 1998a).

Fig. 5.

Reaction mechanism for P. haloplanktis α-amylase (AHA) and chloride-dependent α-amylases in general. By analogy with other studies of glycosidases, Asp 174 has been proven to be the catalytic nucleophile (McCarter and Withers 1996), whereas Glu200 is the best candidate for being the proton donor (Svensson and Søgaard 1993). The role of the third catalytic residue, Asp264, is more unclear, but it has been proposed that it stabilizes the protonated state of the glutamic side-chain (Strokopytov et al. 1995) and that it may contribute to control and/or maintain an elevated pK_a value of the nearby proton donor (Brzozowski and Davies 1997). These studies propose another function of Asp264: In conjunction with the chloride ion, it creates a negative field that forces the water molecules implicated in hydrolysis (after being deprotonated by Glu200) to move, because of electrostatic repulsion, toward the anomeric C1 atom, where it attacks the substrate.