Functional interaction between the N- and C-terminal halves of human hexokinase II

Hossein Ardehali, Richard L. Printz, Richard R. Whitesell, James M. May, Daryl K. Granner*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Mammalian hexokinases (HKs) I-III are composed of two highly homologous ~50-kDa halves. Studies of HKI indicate that the C-terminal half of the molecule is active and is sensitive to inhibition by glucose 6-phosphate (G6P), whereas the N-terminal half binds G6P but is devoid of catalytic activity. In contrast, both the N- and C-terminal halves of HKII (N-HKII and C-HKII, respectively) are catalytically active, and when expressed as discrete proteins both are inhibited by G6P. However, C-HKII has a significantly higher K(i) for G6P (K(iG6P)) than N-HKII. We here address the question of whether the high K(iG6P) of the C-terminal half (C-half) of HKII is decreased by interaction with the N-terminal half (N-half) in the context of the intact enzyme. A chimeric protein consisting of the N-half of HKI and the C-half of HKII was prepared. Because the N-half of HKI is unable to phosphorylate glucose, the catalytic activity of this chimeric enzyme depends entirely on the C-HKII component. The K(iG6P) of this chimeric enzyme is similar to that of HKI and is significantly lower than that of C-HKII. When a conserved amino acid (Asp209) required for glucose binding is mutated in the N-half of this chimeric protein, a significantly higher K(iG6P) (similar to that of C-HKII) is observed. However, mutation of a second conserved amino acid (Ser155), also involved in catalysis but not required for glucose binding, does not increase the K(iG6P) of the chimeric enzyme. This resembles the behavior of HKII, in which a D209A mutation results in an increase in the K(iG6P) of the enzyme, whereas a S155A mutation does not. These results suggest an interaction in which glucose binding by the N-half causes the activity of the C-half to be regulated by significantly lower concentrations of G6P.

Original languageEnglish (US)
Pages (from-to)15986-15989
Number of pages4
JournalJournal of Biological Chemistry
Volume274
Issue number23
DOIs
StatePublished - Jun 4 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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