Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution.

Hexokinase I governs the rate-limiting step of glycolysis in brain tissue, being inhibited by its product, glucose 6-phosphate, and allosterically relieved 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 and 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 directed mutation to block dimerization, remains monomeric at high protein concentration under all conditions of ligation. This nondimerizing mutant exhibits wild-type activity, potent inhibition by glucose 6-phosphate, and phosphate reversal of product inhibition. Small-angle X-ray scattering data from the mutant hexokinase I in the presence of glucose/phosphate, glucose/glucose 6-phosphate, and glucose/ADP/Mg2+/AlF3 are consistent with a rodlike conformation for the monomer similar to that observed in crystal structures of the hexokinase I dimer. Hence, any mechanism for allosteric regulation of hexokinase I should maintain a global conformation of the polypeptide similar to that observed in crystallographic structures.