Hexokinase catalyzes the ATP-dependent phosphorylation of a broad spectrum of 6-carbon sugars. In the reaction involving glucose and ATP, glucose-6-phosphate (G6P) and ADP are formed.

D-Glucose + ATP <=> G6P + ADP + H⁺ 

(see ). This reaction constitutes the first step of the process of glycolysis and is energetically favorable ( = -16.7 kJ/mol).

Hexokinase requires Mg²⁺. The enzyme has a low KM for the sugar substrate ( about 0.1 mM). Hexokinase is inhibited by the product of its reaction, G6P.

Intracellular levels of glucose are usually far higher than the KM value for hexokinase, meaning that the enzyme functions in vivo at saturating substrate concentrations. A liver form of the enzyme called glucokinase has the same substrate specificity, but a significantly higher KM (about 10mM).

Hexokinase is not involved in the process of gluconeogenesis. Instead, the enzyme glucose-6-phosphatase catalyzes the hydrolysis of G6P to glucose and Pi, bypassing the large of the hexokinase-catalyzed reaction.

Next step of glycolysis

1. Rasmol Image of Hexokinase (slow)

2. Glycolysis/Gluconeogenesis