TY - JOUR
T1 - Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation
AU - Li, L.
AU - Lin, K.
AU - Kurland, I. J.
AU - Correia, J. J.
AU - Pilkis, S. J.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1992
Y1 - 1992
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 homogeneity 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 Arg195Ala 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-biphosphatase. 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.
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 homogeneity 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 Arg195Ala 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-biphosphatase. 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.
UR - http://www.scopus.com/inward/record.url?scp=0026730248&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026730248&partnerID=8YFLogxK
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 -