Abstract
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 Km values for fructose 2,6-bisphosphate, Ki 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 Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka 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 Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238 → Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4386-4393 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 267 |
| Issue number | 7 |
| State | Published - Mar 5 1992 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase : Mutation at the fructose 6-phosphate binding site affects phosphate activation. / Li, Lin; Lin, Kai; Kurland, Irwin J.; Correia, John J.; Pilkis, Simon J.
In: Journal of Biological Chemistry, Vol. 267, No. 7, 05.03.1992, p. 4386-4393.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase
T2 - Mutation at the fructose 6-phosphate binding site affects phosphate activation
AU - Li, Lin
AU - Lin, Kai
AU - Kurland, Irwin J.
AU - Correia, John J.
AU - Pilkis, Simon J.
PY - 1992/3/5
Y1 - 1992/3/5
N2 - 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 Km values for fructose 2,6-bisphosphate, Ki 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 Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka 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 Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238 → Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km 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.
AB - 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 Km values for fructose 2,6-bisphosphate, Ki 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 Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka 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 Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238 → Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km 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.
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M3 - Article
C2 - 1311308
AN - SCOPUS:0026730248
VL - 267
SP - 4386
EP - 4393
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 7
ER -