The Effect of Potassium on the Structural Flexibility of β‐amylase2

Abstract

Starch is an important compound in the lives of both plants and humans, with roles in human nutrition and industry such as paper making. β‐amylases are a class of proteins that break down starch in plants to produce maltose. My focus of study is β‐amylase2 (BAM2); one of the β‐amylase genes Arabidopsis thaliana. While the physiological function of BAM2 is unclear, we know three key things about its properties: 1) BAM2 has a tetrameric structure, 2) the enzyme exhibits sigmoidal kinetics through secondary binding sites, and 3) potassium salts are required for catalytic activity. We investigated the molecular basis for potassium stimulating activity in BAM2. Using molecular dynamics, we simulated the effect of potassium on BAM2 finding that potassium increased the flexibility of the enzyme. We are purifying BAM2 wild type and BAM2 with point substitutions which we propose alter the effect of potassium activity. Using these enzyme variants, we compared the structural stability of the enzymes in the presence of potassium, lithium, and sodium using Differential Scanning Fluorimetry (DSF) and Circular Dichroism (CD). Results from DSF showed that the melting temperature decreased with increasing salt concentration, which we interpret as an increase in structural flexibility of BAM2. The CD results support this trend as the melting temperature for BAM2 in the presence of lithium is higher than that of BAM2 in the presence of potassium. These data support a mechanism where potassium ions enhance the flexibility of BAM2.