ADP-Glucose pyrophosphorylase from potato tubers. Site-directed mutagenesis studies of the regulatory sites

Abstract

Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107-24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg2+. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.

Figure 1

Synthetic oligonucleotides used for site-directed mutagenesis. Underlined nucleotides denote differences from the wild-type sequence. Asterisks indicate strands produced from the phage (M13 mp18 and M13 mp19 for the small and large subunit genes, respectively) and used as templates on the mutagenesis protocol.

Figure 2

The effect of 3-PGA concentration on ADP-Glc PPase activity in the pyrophosphorolysis direction. For the wild-type enzyme and the mutants L_K417AS_wt and L_wtS_K404A, 100% activity represents 42, 12, and 5.2 μmol of ATP produced min⁻¹ mg⁻¹, respectively.