| Human hexokinase I catalyzes the first step in glycolysis, playing a key role in the energy metabolism of brain tissue. The chapters included in this thesis focus on structure-function relationships of hexokinase I. Glucose 6-phosphate (G6P), the reaction product, binds to hexokinase I with apparent negative cooperativity and inhibits catalysis (Chapter II). Both N- and C-terminal binding sites for G6P are functional, yet only one G6P molecule binds to hexokinase I.;A series of site-directed mutations were constructed in the base- and ribose-binding pocket of ATP in C-terminal half of hexokinase I G6P inhibition completely (Chapter III). The same mutations in the context of a truncated form of hexokinase I, which contains only the C-terminal half of the enzyme (mini-hexokinase), do not affect G6P inhibition. Obviously, the effects of the same mutations in full-length and mini-hexokinase I are different. On the basis of these results and structural information, we propose a model for allosteric G6P inhibition of hexokinase I. G6P binds to N-terminal half that reaches the active site of the C-terminal half. Furthermore, the results here suggest that the base- and ribose-binding pocket is the target of allosteric inhibition.;Chapter IV investigates the conformation of hexokinase I under different ligation states. The interface mutant engineered for the investigation is proved to be monomeric by small angel x-ray scattering and x-ray crystallography. The monomeric interface mutant and wild-type hexokinase I have essentially identical kinetic properties, thus evidently dimerization plays no role in hexokinase I function under conditions of in vitro. Small angle x-ray scattering data are consistent with the retention of a rod-like conformation under different ligation states, suggesting only subtle conformational changes in response to different ligands.;Chapter V reveals the ADP binding site in the C-terminal half of hexokinase I. Also, it suggests the location of allosteric interface between the N- and C-halves. Combining the structural information and kinetic results, the authors propose molecular mechanism of allosteric inhibition. |
