Glucose (Glc) may be considered the central metabolite in almost all organisms.  Energy and precursors for the biosynthesis of many cellular components are generated from Glc through the glycolytic pathway; on the other hand, Glc can also be stored for later use, as starch or glycogen. The first step in glycolysis is the phosphorylation of Glc; this keeps Glc into the cell and favors the diffusion of extracellular Glc into the cell by reducing its intracellular concentration.  The difference of cellular concentration between substrates and products makes the phosphorylation of Glc very favorable (DG= -33kJ/mol); as a result, this reaction constitutes an important site for regulation.  Hexokinase (ATP: D-hexose 6-phospho-transferase, EC 2.7.1.1.) is the enzyme responsible for the catalysis of this reaction, exerting a key regulatory role in glycolysis.  Although this enzyme has the ability to phosphorylate different hexoses, phosphorilation of Glc is, by far, its most important function.  Four different isozymes of hexokinase have been identified in mammals -Type I, Type II, Type III, and Type IV.  As the latter isozyme has a strong specificity for D-Glc, it is also known as glucokinase.

The structure of Types I-III isozymes consists of two globular halves -the N-terminal and C-terminal regions- held together by a connecting helix and a few hydrogen bonds (100 kDa).  The structure of each half is similar to that of yeast hexokinase, which consists of two regions, i.e. the large and the small lobe; the glucose binding site is located in the bottom of the cleft between the two lobes. These three types of mammalian hexokinases appear to have evolved from an ancestral hexokinase of 50 kDa, by gene duplication and fusion; consequently, they have extensive sequence repetition, both between them and between their N-terminal and C-terminal regions.  A common feature of these isozymes, also present in the ancestral hexokinase, is the regulatory role of Glc phosphorylation through inhibition by the product (glucose-6-phosphate, Glc-6-P).  However, each isozyme displays particular properties and differs from the others in its tissue specificity.  For example, while Type II isozyme possesses catalytic activity in both halves, Type I and Type III isozymes have lost the catalytic activity of the N-terminal region. 

Hexokinase I exhibits a unique regulatory property: physiological levels of inorganic phosphate (Pi) can reverse the inhibition of Glc phosphorylation effected by Glc-6-P.  This characteristic defines hexokinase I as an enzyme with a catabolic role, while the other hexokinases are considered to have mainly an anabolic role.  This supports the fact that hexokinase I is particularly abundant in highly energy-demanding cells, as brain and red blood cells.