Mutagenesis of the glucose-1-phosphate-binding site of potato tuber ADP-glucose pyrophosphorylase

Abstract

Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg2+ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, SK198RLK213R, was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate.

Figure 1

Nucleotide sequence and encoded protein sequence of the potato tuber ADPGlc PPase gene in the region of Lys-198 in the small subunit and Lys-213 in the large subunit. The synthetic oligonucleotides used for site-directed mutagenesis at these positions are shown beside the corresponding mutants they created. The codons for position 198 in the small subunit and the anticodons for position 213 in the large subunit are underlined.

Figure 2

Glc-1-P dependence for wild-type and mutant enzymes. A, For wild-type (•), S_K198RL_wt(▴), S_K198AL_wt(▪), and S_K198EL_wt(○), 100% activity corresponds to 1.2, 16.6, 33.7, and 39.0 nmol 10 min⁻¹, respectively. B, For wild-type (•) and S_wtL_K213R (□), 100% activity corresponds to 1.2 and 1.6 nmol 10 min⁻¹, respectively. Initial velocities of the enzymes were determined in the ADPGlc-synthesis direction (assay II), as described in “Materials and Methods,” with the concentration of Glc-1-P being varied. The amounts of the wild-type, S_K198RL_wt, S_K198AL_wt, S_K198EL_wt, and S_wtL_K213R proteins were about 2.5 × 10⁻³, 70 × 10⁻³, 80 × 10⁻³, 2.4 × 10⁻³, and 5.5 × 10⁻³ μg, respectively.