The Importance of a Loop Structure in ATP Apparent Affinity and Allosteric Activation of Escherichia coli ADP‐Glucose Pyrophosphorylase

Abstract

Previous work into the study Escherichia coli ADP‐Glc PPase has identified several regions of the N‐terminal domain that appear to play a key role in the allosteric activation of the enzyme by Fructose‐1,6‐bisphosphate (FBP). One such region involves a loop structure formed by residues Pro103‐Gln116. Alanine scanning mutagenesis was performed on residues of this loop. The results of kinetic assays monitoring the forward (ADP‐Glc synthesis) reaction show that several residues play either a direct or indirect role in ATP binding or activation of the enzyme by FBP. These are Pro103, Gln105, Gln106, Arg107, Trp113, Tyr114, and Arg115. Aside from Arg115, whose main effect appears to have more to do with ATP binding than FBP activation, the defining characteristic of the alanine mutants was the failure to be appreciably activated by Fru‐1,6‐BP. These mutants saw near wild type activity in the absence of FBP but were only activated 1.5‐ to 5‐fold by FBP compared to 30‐fold for the wild type. Using a homology model of the E. coli enzyme (based on the crystal structures of closely related ADP‐Glc PPases from Agrobacterium tumefaciens and Potato tuber), we hypothesize some structure‐function relationships. For instance, the importance Gln105 and Gln106can likely be associated with hydrogen bond interactions with another loop structure formed by Tyr75‐Gln78 which has previously been identified as being important for ATP binding. We conclude that most of the loop is important to trigger the activation by FBP and increase the apparent affinity for ATP. Supported by funds from NSF MCB‐1024945