Identification of Functionally Important Amino Terminal Arginines of Agrobacterium tumefaciens ADPglucose Pyrophosphorylase by Alanine Scanning Mutagenesis

D.F. GÓMEZ CASATI, R.Y. IGARASHI, C.N. BERGER, M.E. BRANDT, A.A. IGLESIAS Y C.R. MEYER

BIOCHEMISTRY

American Chemical Society

Año: 2001 vol. 40 p. 10169 - 10178

0006-2960

ABSTRACT: Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase with the arginylreagent 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.