Hexose Phosphate Binding Sites of Fructose 6-Phosphate, 2-Kinase:Fructose 2,6-Bisphosphatase

Tomoyuki Tsujikawa, Fusao Watanabe, Kosaku Uyeda

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

A previous chemical modification study [Kitamura et al. (1989) J. Biol. Chem. 264, 6344-6438] has shown that yV-bromoacetylethanolamine phosphate labeled specifically Cysl07 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 LyslOO, Lysl03, and Aspll2 caused at most a 2-fold increase in KmF6P and a 2-3-fold increase in KmF6P, suggesting that these residues are not involved in the direct binding of Fru 6-P. However, change of Argl02 to Leu and to Lys resulted in a 325 × and 22 ×, respectively, increase in KmF6P, and change of Argl02 to Glu resulted in nearly complete loss of the kinase activity. Change of Cys 105 to Ala or Ser increased ATmF6P about 10 ×. The Vmax of all these mutated enzymes except the one that changed Argl02 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 Argl02 (R102K) were slightly more susceptible to guanidine inactivation than the wild-type enzyme. These results suggest that Argl02 is essential for binding the 6-phosphate of Fru 6-P and that Cys 105 also may play a role at the same site.

Original languageEnglish (US)
Pages (from-to)6389-6393
Number of pages5
JournalBiochemistry
Volume34
Issue number19
DOIs
StatePublished - May 1 1995

ASJC Scopus subject areas

  • Biochemistry

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