Site-Directed Mutagenesis of a Regulatory Site ofEscherichia coliADP-Glucose Pyrophosphorylase: The Role of Residue 336 in Allosteric Behavior

Site-directed mutagenesis was used to probe the role of glycine residue 336 in the regulatory properties ofEscherichia coliADP-glucose pyrophosphorylase. This residue was previously found to be changed from glycine to aspartate in the gene of anEscherichia colimutant strain. The mutant enzyme had altered kinetic properties, including higher activity in the absence of the activator fructose 1,6-bisphosphate (FBP), higher apparent affinity for FBP and substrates, and lower apparent affinity for the inhibitor AMP. The observed changes in activity were caused by this single mutation, because the aspartate mutant was prepared from the wild-type gene. The kinetic properties of the site-directed mutant are identical to those of the enzyme from the mutant strain. A series of mutants was prepared to explore the effects of charge, size, shape, and hydrophobicity of the amino acid at residue 336 on the enzyme regulatory properties. All of the mutants, except for the lysine and arginine enzymes, were expressed and purified for kinetic analysis. The glycine-336 residue is able to tolerate diverse substitutions without compromise of catalytic activity. A range of allosteric changes was observed, with the most dramatic effects seen with the highly active aspartate enzyme and the low-activity G336Q mutant, which exhibited lower apparent affinities for activator and substrates and higher apparent affinity for inhibitor. The altered allosteric properties of the G336D mutant enzyme were almost completely abolished by substitution of asparagine. Thus, the aspartate negative charge is essential for the altered binding of effectors.