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.