Hexokinase (HK) Enzyme Commission (EC) Number 2.7.1.1

Propriedades

Descrição

General description

ATP:D-hexose 6-phosphotransferase

Hexokinase consists of 486 amino acids and has a molecular weight of 57,000 D (SDS-PAGE) in citrate/phosphate buffer. It may form dimers under other buffer conditions.

Hexokinase exists in three isozymes, namely type I, type II and type III that differ in subcellular localization, catalytic and regulatory properties. The type I isozyme serves a catabolic function facilitating the entry of glucose into glycolytic metabolism for energy production. The type II and type III enzymes perform anabolic functions, such as generating G6P for glycogen synthesis or metabolism through the phosphogluconate pathway for lipid synthesis. Type I and type II isozymes have been found to be bound to mitochondria. The type III isozyme has been found to have perinuclear localization in several cell types.^[6]

Application

Hexokinase (HK) has been used in enzyme assays^[4][7] and chemiluminescence assay.^[1] It has also been used to determine ATP concentration.^[8]

Use Hexokinase for the determination of D-glucose^[3][4], D-fructose, and D-sorbitol in food or biological research samples. Hexokinase may also be used for the assay of other saccharides, which are convertible to glucose or fructose, and is, therefore, useful in the assay of many glycosides.
Hexokinase has been used in the enzymatic detection of nitric oxide^[1] and in the measurement of ATP by luminescence method.^[2]

Biochem/physiol Actions

Hexokinase catalyzes the conversion of hexoses to hexose-6-phosphate in an ATP-dependent manner. It is also called as the glycolytic enzyme as it catalyzes the first step in glucose metabolism with the formation of glucose-6-phosphate (G6P). In eukaryotes, it functions as a glucose sensor.^[5]

Quality

Contaminants: <0.002% PGI, <0.0005% G6P-DH and GR, each, <0.001% CK, MK, 6-PGDH, and ADH, each, <0.05% GIDH and invertase (β-fructosidase), each, <30 μg/ml glucose (enzymatically)

Unit Definition

Unit Assay: For measuring hexokinase activity 0.22 M glucose in 0.1 M triethanolamine buffer, pH 7.6, 6.5 mM MgCI₂, 2.7 mM ATP, 0.83 mM NADP is incubated in the presence of 1.7 U glucose-6-phosphate dehydrogenase with an appropriate amount of hexokinase (10–20 mU) at +25 °C (total volume: 3.0 ml). The enzyme activity is calculated from the increase of absorbance at 340 nm ( = 6.3 [I × mmol/l^-1 × cm-1]) or 365 nm ( = 3.5 [I × mmol/l^-1 × cm^-1]).

Physical form

Suspension in 3.2 M ammonium sulfate solution, pH approximately 6.5

Preparation Note

Activator: Hexokinase requires Mg²⁺ ions (K_m = 2.6 mM) for activity. Catecholamines too increase activity.
Stabilizers: Thiols