PURIFICATION AND CHARACTERIZATION OF THE CARBOXYL-DOMAIN OF HUMAN HEXOKINASE TYPE-III EXPRESSED AS FUSION PROTEIN

In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These p roteins are homologous and are organized in two homologous domains, wi th the exception of hexokinase type IV which has only one. This organi zation is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In th is study we cloned the carboxyl-domain of human hexokinase type III an d expressed it in Escherichia coli as a glutathione S-transferase fusi on protein, using the pGEX-2T expression vector. The recombinant prote in showed catalytic activity. A comparative study of the kinetic prope rties of the expressed carboxyl-domain and the enzyme partially purifi ed from human lymphocytes is also shown. The results now allow a bette r understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.