Evidence for Specific Roles of Arginines 26 and 38 in the Allosteric Regulation of ADP Glucose Pyrophosphorylase from Thermus thermophilus

Abstract

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosteric enzyme that catalyzes the rate‐limiting step in glucan synthesis, an attractive target for engineering to increase the production of renewable carbon. The thermophillic bacterial enzyme from Thermus thermophilus was found to be activated by G6P, F6P, and FBP and displayed optimal activity at 75°C. Based on alignment and molecular modeling studies, R26 and R38 were proposed to be invovled in allosteric regulation of this enzyme. Altered proteins (R26A and R38A) were generated, expressed, and purified. R26A and R38A enzymes displayed a significant decrease in Vmax and a decrease in the apparent affinity for substrates compared to the wild‐type enzyme. In contrast to the wild‐type, the presence of activators caused an increased apparent affinity for the substrate ATP for both enzymes. The R26A mutation resulted in an ~8‐fold decrease in apparent affinity for G6P at 37°C, and a ~30‐fold decrease at 75°C. The R38A enzyme showed ~6‐fold and ~8‐fold decreases in apparent affinity for G6P and F6P at 37°C, respectively. Preliminary data shows decreased apparent affinity for substrates at 75°C for both enzymes. The diminution of the Vmax is consistent with a role for these arginines in stabilizing an active conformation of the enzyme. In addition, R26 appears to play a role in the binding of G6P while R38 appears to facilitate binding of both G6P and F6P in the allosteric subsite(s). Complete kinetic characterization of the altered enzymes and double mutant R26A, R38A at 75°C is underway. Supported by NSF Award 0448676.