THE ROLES OF GLYCINE RESIDUES IN THE ATP BINDING-SITE OF HUMAN BRAIN HEXOKINASE

Mutants of hexokinase I (Arg(539) --> Lys, Thr(661) --> Ala, Thr(661) --> Val, Gly(534) --> Ala, Gly(679) --> Ala, and Gly(862) --> Ala), lo cated putatively in the vicinity of the ATP binding pocket, were const ructed, purified to homogeneity, and studied by circular dichroism (CD ) spectroscopy, fluorescence spectroscopy, and initial velocity kineti cs, The wild-type and mutant enzymes have similar secondary structures on the basis of CD spectroscopy, The mutation Gly(679) --> Ala had li ttle effect on the kinetic properties of the enzyme, Compared with the wild type enzyme, however, the Gly(534) --> Ala mutant exhibited a 40 00-fold decrease in k(cat) and the Gly(862) --> Ala mutant showed an 1 1-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, I norganic phosphate is, however, less effective in relieving glucose 6- phosphate inhibition of the Gly(862) --> Ala mutant, relative to the w ild-type enzyme and entirely ineffective in relieving inhibition of th e Gly(534) --> Ala mutant. Although the fluorescence emission spectra showed some difference for the Gly(862) --> Ala mutant relative to tha t of the wild-type enzyme, indicating an environmental alteration arou nd tryptophan residues, no change was observed for the Gly(534) --> Al a and Gly(679) --> Ala mutants, Gly(862) --> Ala and Gly(534) --> Ala are the first instances of single residue mutations in hexokinase I th at affect the binding affinity of ATP and abolish phosphate-induced re lief of glucose 6-phosphate inhibition, respectively.