The roles of glycine residues in the ATP binding site of human brain hexokinase.
Mutants of hexokinase I (Arg539 --> Lys, Thr661 --> Ala, Thr661 --> Val, Gly534 --> Ala, Gly679 --> Ala, and Gly862 --> Ala), located putatively in the vicinity of the ATP binding pocket, were constructed, purified to homogeneity, and studied by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and initial velocity kinetics. The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy. The mutation Gly679 --> Ala had little effect on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly534 --> Ala mutant exhibited a 4000-fold decrease in kcat and the Gly862 --> Ala mutant showed an 11-fold increase in Km for ATP. Glucose 6-phosphate inhibition of the three glycine mutants is comparable to that of the wild-type enzyme. Inorganic phosphate is, however, less effective in relieving glucose 6-phosphate inhibition of the Gly862 --> Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly534 --> Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly862 --> Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly534 --> Ala and Gly679 --> Ala mutants. Gly862 --> Ala and Gly534 --> Ala are the first instances of single residue mutations in hexokinase I that affect the binding affinity of ATP and abolish phosphate-induced relief of glucose 6-phosphate inhibition, respectively.
Zeng
C
C
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.
Aleshin
A E
AE
Chen
G
G
Honzatko
R B
RB
Fromm
H J
HJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002942:Circular Dichroism; D005998:Glycine / Q000378:metabolism*; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010710:Phosphates / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D010710:Phosphates; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005998:Glycine