Structural and functional aspects of chloride binding to Alteromonas haloplanctis alpha-amylase.
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.
Feller
G
G
Laboratory of Biochemistry, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium.
Bussy
O
O
Houssier
C
C
Gerday
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002414:Cations, Monovalent; D002712:Chlorides / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007555:Isoxazoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry*
D001426:Bacterial Proteins; D002414:Cations, Monovalent; D002712:Chlorides; D004791:Enzyme Inhibitors; D007555:Isoxazoles; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D000516:alpha-Amylases