Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase: Mutation at the fructose 6-phosphate binding site affects phosphate activation

To identify those residues involved in fructose 6-phosphate binding to the kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase site-directed mutations were engineered at Lys¹⁹⁴, Arg¹⁹⁵, Arg²³⁰, and Arg²³⁸. The mutant enzymes were purified to homogeniety by anion exchange and Blue-Sepharose chromatography and/or substrate elution from phosphocellulose columns. Circular dichroism experiments demonstrated that all of the single amino acid mutations had no effect on the secondary structure of the protein. In addition, when fructose-2,6-bisphosphatase activity was measured, all mutants had K_m values for fructose 2,6-bisphosphate, K_i values for fructose 6-phosphate, and maximal velocities similar to that of the wild-type enzyme. Mutation of Arg¹⁹⁵ → Ala, or His, had little or no effect on the maximal velocity of the kinase but increased the K_m for fructose 6-phosphate greater than 3,000-fold. Furthermore, the K_a for phosphate for Arg 195Ala was increased 100-fold compared with the wild-type enzyme. Mutation of Lys¹⁹⁴ → Ala had no effect on maximal velocity or the K_m for fructose 6-phosphate. Mutation of either Arg²³⁰ or Arg²³⁸ → Ala increased the maximal velocity and the K_m for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the K_m values for ATP of the Arg230Ala and Arg238Ala mutants were 30-40-fold higher than that for the wild-type enzyme. Mutation of Gly⁴⁸ → Ala resulted in a form with no kinase activity, but fructose-2, 6-bisphosphatase activity was identical to that of the wild-type enzyme. The results indicate that: 1) Arg¹⁹⁵ is a critical residue for the binding of fructose 6-phosphate to the 6-phosphofructo-2-kinase domain, and that interaction of the sugar phosphate with Arg¹⁹⁵ is highly specific since mutation of the adjacent Lys¹⁹⁴ → Ala had no effect on fructose 6-phosphate binding; 2) Arg¹⁹⁵ also play an important role in the binding of inorganic phosphate; and 3) Gly⁴⁸ is an important residue in the nucleotide binding fold of 6-phosphofructo-2-kinase and that both Arg²³⁰ and Arg²³⁸ are also involved in ATP binding; and 4) the bifunctional enzyme has two separate and independent fructose 6-phosphate binding sites.