The Roles of Glycine Residues in the ATP Binding Site of Human Brain Hexokinase

Chenbo Zeng, Alexander E. Aleshin, Guanjun Chen, Richard B. Honzatko, Herbert J. Fromm
1998 Journal of Biological Chemistry  
Mutants of hexokinase I (Arg 539 3 Lys, Thr 661 3 Ala, Thr 661 3 Val, Gly 534 3 Ala, Gly 679 3 Ala, and Gly 862 3 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 Gly 679 3 Ala had little effect on the kinetic
more » ... on the kinetic properties of the enzyme. Compared with the wild-type enzyme, however, the Gly 534 3 Ala mutant exhibited a 4000-fold decrease in k cat and the Gly 862 3 Ala mutant showed an 11-fold increase in K m 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 Gly 862 3 Ala mutant, relative to the wild-type enzyme and entirely ineffective in relieving inhibition of the Gly 534 3 Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly 862 3 Ala mutant relative to that of the wild-type enzyme, indicating an environmental alteration around tryptophan residues, no change was observed for the Gly 534 3 Ala and Gly 679 3 Ala mutants. Gly 862 3 Ala and Gly 534 3 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.
doi:10.1074/jbc.273.2.700 pmid:9422720 fatcat:c7r2m6waizf5jpjubbwtdgtziy