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

Mammalian hexokinases (HKs) I-III are composed of two highly homologous sim ilar 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-phosphat e (G6P), whereas the N-terminal half binds G6P but is devoid of catalytic a ctivity. In contrast, both the N- and C-terminal halves of HKII (N-HKII and C-HKII, respectively) are catalytically active, and when expressed as disc rete proteins both are inhibited by G6P, However, C-HKII has a significantl y 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 decrease d by interaction with the N-terminal half (N-half) in the context of the in tact enzyme. A chimeric protein consisting of the N-half of HKI and the C-h alf of HKII was prepared. Because the N-half of HKI is unable to phosphoryl ate glucose, the catalytic activity of this chimeric enzyme depends entirel y 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 conserv ed amino acid (Asp(209)) required for glucose binding is mutated in the N-h alf of this chimeric protein, a significantly higher K-iG6P (similar to tha t of C-HKII) is observed. However, mutation of a second conserved amino aci d (Ser(155)), also involved in catalysis but not required for glucose bindi ng, 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 sugg est an interaction in which glucose binding by the N-half causes the activi ty of the C-half to be regulated by significantly lower concentrations of G 6P.