Expression of human brain hexokinase in Escherichiacoli: Purification and characterization of the expressed enzyme

Human brain hexokinase (hexokinase I) was produced in Escherichiacoli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5′ flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an aboundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to nerr homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 μM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl₂. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.