Ae. Aleshin et al., Nonaggregating mutant of recombinant human hexokinase I exhibits wild-typekinetics and rod-like conformations in solution, BIOCHEM, 38(26), 1999, pp. 8359-8366
Hexokinase I governs the rate-limiting step of glycolysis in brain tissue,
being inhibited by its product, glucose 6-phosphate, and allosterically rel
ieved of product inhibition by phosphate. On the basis of small-angle X-ray
scattering, the wild-type enzyme is a monomer in the presence of glucose a
nd phosphate at protein concentrations up to 10 mg/mL, but in the presence
of glucose 6-phosphate, is a dimer down to protein concentrations as low as
1 mg/mL. A mutant form of hexokinase I, specifically engineered by directe
d mutation to block dimerization, remains monomeric at high protein concent
ration under all conditions of ligation. This nondimerizing mutant exhibits
wild-type activity, potent inhibition by glucose 6-phosphate, and phosphat
e reversal of product inhibition. Small-angle X-ray scattering data from th
e mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose
6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike confo
rmation for the monomer similar to that observed in crystal structures of t
he hexokinase I dimer. Hence, any mechanism for allosteric regulation of he
xokinase I should maintain a global conformation of the polypeptide similar
to that observed in crystallographic structures.