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.
Aleshin
A E
AE
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames 50011, USA.
Malfois
M
M
Liu
X
X
Kim
C S
CS
Fromm
H J
HJ
Honzatko
R B
RB
Koch
M H
MH
Svergun
D I
DI
D001921:Brain; D002942:Circular Dichroism; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D006593:Hexokinase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D012996:Solutions; D014965:X-Rays
D011994:Recombinant Proteins; D012996:Solutions; D006593:Hexokinase