STRUCTURAL AND FUNCTIONAL-ASPECTS OF CHLORIDE BINDING TO ALTEROMONAS-HALOPLANCTIS ALPHA-AMYLASE

Chloride is the allosteric effector of vertebrate pancreatic and saliv ary 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 restric ted to that of Cl-, is essential for the amylolytic reaction, Engineer ing of the chloride binding site reveals that a basic residue is an es sential component of the site, The mutation K337R alters the Cl--bindi ng properties, whereas the mutation K337Q produces an active, chloride -independent enzyme. Comparison of the K-d values for Cl- in three hom ologous alpha-amylases also indicates that the binding affinity is dep endent on the chloride coordination mode by this basic residue. Analys is of substrate and chloride binding according to the allosteric kinet ic model shows that the chloride effector is not involved in substrate binding, By contrast, the pH dependence of activity and experiments o f chemical modifications and Ca2+ inhibition show that the chloride io n is responsible for the pK(alpha) shift of catalytic groups and inter acts with active site carboxyl groups.