Identification of Functionally Important Amino-Terminal Arginines of Agrobacterium tumefaciens ADP-Glucose Pyrophosphorylase by Alanine Scanning Mutagenesis
Citations Over Time
Abstract
Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase with the arginyl reagent phenylglyoxal resulted in complete desensitization to fructose 6-phosphate (F6P) activation, and partial desensitization to pyruvate activation. The enzyme was protected from desensitization by ATP, F6P, pyruvate, and phosphate. Alignment studies revealed that this enzyme contains arginine residues in the amino-terminal region that are relatively conserved in similarly regulated ADP-glucose pyrophosphorylases. To functionally evaluate the role(s) of these arginines, alanine scanning mutagenesis was performed to generate the following enzymes: R5A, R11A, R22A, R25A, R32A, R33A, R45A, and R60A. All of the enzymes, except R60A, were successfully expressed and purified to near homogeneity. Both the R5A and R11A enzymes displayed desensitization to pyruvate, partial activation by F6P, and increased sensitivity to phosphate inhibition. Both the R22A and R25A enzymes exhibited reduced Vmax values in the absence of activators, lower apparent affinities for ATP and F6P, and reduced sensitivities to phosphate. The presence of F6P restored R22A enzyme activity, while the R25A enzyme exhibited only ∼1.5% of the wild-type activity. The R32A enzyme displayed an ∼11.5-fold reduced affinity for F6P while exhibiting behavior identical to that of the wild type with respect to pyruvate activation. Both the R33A and R45A enzymes demonstrated a higher activity than the wild-type enzyme in the absence of activators, no response to F6P, partial activation by pyruvate, and desensitization to phosphate inhibition. These altered enzymes were also insensitive to phenylglyoxal. The data demonstrate unique functional roles for these arginines and the presence of separate subsites for the activators.
Related Papers
- → Alanine Scanning Effects on the Biochemical and Biophysical Properties of Intrinsically Disordered Proteins: A Case Study of the Histidine to Alanine Mutations in Amyloid-β42(2019)35 cited
- → An essential arginine residue in porcine phospholipiase A2.(1980)32 cited
- → Inhibition of the lepidopteran amino acid co‐transporter KAAT1 by phenylglyoxal: role of arginine 76(2002)9 cited
- → Deep Alanine Scanning Reveals Potent Multi-alanine-substituted Protein–protein Interaction Inhibitors(2021)1 cited
- → Arginine Appears not to be Located in the Active Site of Na+/K+-ATPase(1994)