Hexose phosphate binding sites of fructose 6-phosphate,2-kinase

Fructose 2,6-bisphosphatase

Tomoyuki Tsujikawa, Fusao Watanabe, Kosaku Uyeda

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

A previous chemical modification study [Kitamura et al. (1989) J. Biol. Chem. 264, 6344-6438] has shown that N-bromoacetylethanolamine phosphate labeled specifically Cys107 of rat liver Fru 6-P,2-kinase:Fru 2,6-Pase and the corresponding Cys of the bovine heart enzyme, leading to inactivation of kinase activity. Since Fru 6-P provided protection against the inactivation, this region of the enzyme was thought to be a Fru 6-P binding site of the kinase enzyme. To examine this possibility, oligonucleotide-directed mutagenesis has been used to alter several residues in expressed rat testis Fru 6-P,2-kinase:Fru 2,6-Pase. The change of Lys100, Lys103, and Asp112 caused at most a 2-fold increase in Km F6P and a 2-3-fold increase in Km ATP, suggesting that these residues are not involved in the direct binding of Fru 6-P. However, change of Arg102 to Leu and to Lys resulted in a 325x and 22x, respectively, increase in Km F6P, and change of Arg102 to Glu resulted in nearly complete loss of the kinase activity. Change of Cys105 to Ala or Ser increased Km F6P about 10x. The Vmax of all these mutated enzymes except the one that changed Arg102 to Glu (R102E) was increased 10% to 85%. The kinetic parameters of Fru 2,6-Pase were not altered by these changes. R102E formed several polymeric forms of the enzyme, including a tetramer. Both R102E and an additional derivative that substituted Lys for Arg102 (R102K) were slightly more susceptible to guanidine inactivation than the wild-type enzyme. These results suggest that Arg102 is essential for binding the 6-phosphate of Fru 6-P and that Cys105 also may play a role at the same site.

Original languageEnglish (US)
Pages (from-to)6389-6393
Number of pages5
JournalBiochemistry®
Volume34
Issue number19
StatePublished - 1995

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Phosphofructokinase-2
Hexoses
Phosphates
Binding Sites
Enzymes
Phosphotransferases
Rats
Mutagenesis
Guanidine
Chemical modification
Site-Directed Mutagenesis
Kinetic parameters
Oligonucleotides
Liver
Testis
Derivatives

ASJC Scopus subject areas

  • Biochemistry

Cite this

Hexose phosphate binding sites of fructose 6-phosphate,2-kinase : Fructose 2,6-bisphosphatase. / Tsujikawa, Tomoyuki; Watanabe, Fusao; Uyeda, Kosaku.

In: Biochemistry®, Vol. 34, No. 19, 1995, p. 6389-6393.

Research output: Contribution to journalArticle

Tsujikawa, Tomoyuki ; Watanabe, Fusao ; Uyeda, Kosaku. / Hexose phosphate binding sites of fructose 6-phosphate,2-kinase : Fructose 2,6-bisphosphatase. In: Biochemistry®. 1995 ; Vol. 34, No. 19. pp. 6389-6393.
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