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

Mammalian hexokinases (HKs) I-III are composed of two highly homologous similar to 50-kDa halves. Studies of HKI indicate that the C-terminal half of the molecule is active and is sensitive to inhibition by glucose B-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 (Asp(209)) 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 (Ser(155)), 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.