This protein is classified as a homodimer, consisting of two similar subunits connected via α-helix (residues 100-109).  Each monomer contains two domains – a small α/β domain, and a larger α + β (separated secondary structures) domain.  The active sites of the enzyme are located at the interface between the large and small domains, indicated by the deep clefts in the tertiary structure of the enzyme at these locations.  There are a total of 332 residues per monomer, for a grand total of 664 residues in the entire protein.  The total secondary structure components for each monomer total 18 α-helices and 13 β-sheets.

The protein consists of a mainly polar shell with a hydrophobic core.  This affirms its placement in the cytosol of E. coli.  The active site pocket appears to sit just beneath the surface of the polar shell, in a slightly hydrophobic pocket containing few polar residues.  The glucose ligands are situated directly between the two large β-sheet components of each monomer.  The active site pockets consist mostly of β-sheet structure.  A large portion of each monomer (e.g. the large domains) consists of α-helices exclusively.  Glucose forms hydrogen bonds with active site residues Asn99, Asp100, Glu157, His160, and Glu187.  Of these residues, only the His160 is not conserved in human hexokinase I.

The proposed catalytic mechanism for this enzyme consists of Asp100 accepting a proton from the O6 hydroxyl group of the glucose molecule.  This is prior to nucleophilic attack of the ATP molecule, which precedes formation of glucose-6-phosphate.