Structural and Functional Aspects of Chloride Binding to Alteromonas haloplanctis α-Amylase

Abstract

Chloride is the allosteric effector of vertebrate pancreatic and salivary alpha-amylases and of the bacterial alpha-amylase from Alteromonas haloplanctis. Activation experiments of A. haloplanctis alpha-amylase by several monovalent anions show that a negative charge, not restricted to that of Cl-, is essential for the amylolytic reaction. Engineering of the chloride binding site reveals that a basic residue is an essential component of the site. The mutation K337R alters the Cl--binding properties, whereas the mutation K337Q produces an active, chloride-independent enzyme. Comparison of the Kd values for Cl- in three homologous alpha-amylases also indicates that the binding affinity is dependent on the chloride coordination mode by this basic residue. Analysis of substrate and chloride binding according to the allosteric kinetic model shows that the chloride effector is not involved in substrate binding. By contrast, the pH dependence of activity and experiments of chemical modifications and Ca2+ inhibition show that the chloride ion is responsible for the pKa shift of catalytic groups and interacts with active site carboxyl groups.

Halorhodopsin pumps Cl–and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

“…Cl -binding in the HR ground state is dependent on both the pH and Cl -concentration, as found in other ion-binding systems (9,10). At low Clconcentrations, no Cl -is bound and the HR absorption spectrum is blue-shifted, indicating that the trans retinal SB is deprotonated (45).…”

“…For example, mutation of a phosphorylation site to an anionic amino acid has been found to yield constitutive activation (7) that can trigger certain cancers (8). Amylases provide another example, where one class is dependent on the pH and Cl -concentration, with an active site Arg or Lys binding the chloride (9,10). In contrast, amylases remove the basic amino acid and have pH-and Cl --independent activity.…”

Group BStreptococcusPullulanase Crystal Structures in the Context of a Novel Strategy for Vaccine Development

“…2 and 4C). Cl Ϫ ions are not present in Pul Bs and Pul Kp ; however, they have been reported in the crystal structures of several ␣-amylases (1,14,24). In fact, several reports describe the identification of Cl Ϫ -dependent ␣-amylases from three gram-negative bacteria, invertebrates, and mammals; such enzymes have been found to also possess additional structural features specific to Cl Ϫ -dependent activity (2, 11).…”

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

“…− definitely leads to a complete loss of the enzymatic activity, which is fully recovered by the incorporation of Cl − and Br − ions into the apo-enzyme (Feller et al 1996). The chloride ion binding site for Cl − -dependent mammalian pancreatic and salivary α-amylases consists of Arg195, Asn298, Arg337, and a water molecule (Qian et al 1993;Brayer et al 1995; 2 P.-L. Ong et al Protein ligands of the chloride-binding site are boxed and are numbered according to the sequence of Bl TreA.…”

“…Notably, the primary difference between the amino acid sequences of Cl − -required α-amylases and those of Cl − -independent enzymes is the K/R337Q/Y substitution. Investigations on Pseudoalteromonas haloplanctis α-amylase have demonstrated that chloride ion is not bound by K337R and the starchdegrading ability of this variant is consequently independent of Cl − (Feller et al 1996). Very recently, we have constructed a chimeric plasmid for the functional expression of Bacillus licheniformis trehalose-6-phosphate hydrolase (Bl TreA) in recombinant Escherichia coli (Chuang et al 2012).…”