Hexokinase and glucokinase enzymes and regulation

Hexokinase and glucokinase enzymes and regulation

Hexokinases Definition:  Group of enzymes that phosphorylates hexoses (six-carbon sugars) forming hexose phosphate.

  • In most organisms, glucose is the most important substrate of hexokinases, and glucose-6-phosphate is the most important product.
  • Hexokinase possesses the ability to transfer an inorganic phosphate group from ATP to a substrate.

Types of mammalian hexokinase

There are four important mammalian hexokinase isozymes encoded by four different genes: Hexokinase I, II, III, and IV (Hexokinase A, B, C and D)

(Note: Isoenzymes or isozymes are different proteins that catalyze the same reaction, and that generally differ in kinetic and regulatory properties, subcellular distribution, or in the cofactors used. They may be present in the same species, in the same tissue or even in the same cell)

Tissue location: Hexokinase I is the predominant isozyme in the brain, whereas in skeletal muscle hexokinase I and II are present.

Hexokinase has different functions in liver and muscle.
Muscle consumes glucose, using it for energy production. Liver maintains blood glucose homeostasis by removing or producing glucose.

Substrate for the enzyme Hexokinase: Any of the hexoses (glucose, fructose mannose etc)

Reaction catalysed: The conversion of glucose to glucose-6-phosphate (G6P)

Glucokinase (hexokinase IV or hexokinase D)

Reaction catalysed: Conversion of glucose into glucose-6-phosphate (G6P).

(This substance isn’t used in glycolysis, but rather forms the glucose polymer glycogen, as well as to fat and cholesterol).

Substrate for the enzyme: The only substrate of glucokinase is D-glucose

Tissue location: Hexokinase IV is mainly present in hepatocytes (Liver cells) and β cells of the pancreas

Gene encoding: The GCK gene on the chromosome 7 in humans.

Comparison of the kinetic properties of hexokinase isozymes

Hexokinase I, II and III

The kinetic properties of hexokinase I, II, and III are similar.

Km value and affinity: Hexokinases I, II, and III are referred to as “low-Km” isozymes (high affinity for glucose – below 1 mM)

Hexokinase I and II have a Km for glucose of 0.03 mM and 0.1 mM, respectively. Therefore these isozymes work very efficiently at normal blood glucose levels 4-5 mM.

Advantage of low Km: Its low Km allows glucose to enter cells, especially brain cells and RBCs, under fasting conditions.

Vmax: Low Vmax (hence saturated in low glucose levels)

Effect of Insulin: No effect on hexokinases

 Hexokinase IV

Km value and affinity: Glucokinase has a high Km value (approximately 10 mM). Hence, it has a lower affinity towards glucose. This means that the enzyme works efficiently only when blood glucose concentration is high, for example after a meal rich in carbohydrates with a high glycemic index.

Advantage of high Km: The low glucose affinity of glucokinase in the liver gives the brain and muscles first call on glucose when its supply is limited, whereas it ensures that glucose will not be wasted when it is abundant.

The high Km also minimizes the uptake of glucose by the liver during fasting, thereby preventing unnecessary synthesis of glycogen and the development of hypoglycemia.

Vmax: high Vmax (The enzyme is only functional at high glucose levels)

The activity of glucokinase depends upon the nutritional state of glucose.

Effect of insulin: Insulin stimulates glucokinase.

(When blood glucose is high insulin is produced, which causes the activation of transcription of the gene that encodes the Glucokinase enzyme in liver).

Figure 1: Comparison of the kinetic properties of hexokinase IV (glucokinase) and hexokinase I.

(When blood glucose rises above 5 mM, hexokinase IV activity increases, but hexokinase I is already operating near Vmax at 5 mM glucose and cannot respond to an increase in glucose concentration).

Hexokinase Isozymes of muscle and liver are differently affected by their product Glucose 6 phosphate

Regulation of the activity of hexokinases I-III (Feed back inhibition)

Hexokinases I-III are allosterically inhibited by glucose 6-phosphate, the product of their reaction. This ensures when glucose is not needed for energy.

High concentrations of this molecule signal that the cell no longer requires glucose for energy. Feedback inhibition ensures that glucose 6-phosphate does not accumulate in the cytosol. As a result excess glucose will be left in the blood which can be used for glycogen synthesis or as a source of biosynthetic precursors. When glucose levels drop, hexokinase activity resumes and thwarts the storage of glucose.

Inhibition of PFK -1 leads to the inhibition of hexokinase

When PFK -1 is inactive, the concentration of fructose 6-phosphate rises. In turn the level of glucose 6-phosphate rises because it is in equilibrium with fructose 6-phosphate. G6P allosterically inhibit  hexokinase enzyme.

Hence, the inhibition of Phosphofructokinase leads to the inhibition of hexokinase.

Regulation of the activity of hepatic glucokinase

Glucokinase differs in three important respects from hexokinases I–III of muscle.

1) The glucose concentration at which hexokinase IV is half saturated (about 10 mM) is higher than the usual concentration of glucose in the blood.

  • The rate at which glucose uptake and phosphorylation occurs in liver are determined by the value of blood glucose level
  • Hepatocytes contain glucose transporter GLUT2, which rapidly equilibrates the glucose concentrations in cytosol and blood.
  • When the blood glucose concentration is high (as it is after a meal rich in carbohydrates) GLUT2 is very active and excess glucose is transported into hepatocytes, where hexokinase IV converts it to glucose 6-phosphate.
  • Due to high Km for glucose, Hexokinase IV is not saturated at 10 mM glucose and its activity continues to increase even when the intracellular concentration of the glucose reaches or exceeds 10 mM.
  • Glucokinase is not subject to product inhibition by glucose-6-phosphate. Liver will take up & phosphorylate glucose even when liver glucose-6-phosphate is high.
  • On the other hand, when glucose availability is low, its concentration in the cytosol of hepatocytes is much lower than the Km for glucose of glucokinase, so that glucose produced through gluconeogenesis and/or glycogenolysis is not phosphorylated and can leave the cell.

2) Hexokinase IV is subject to inhibition by the reversible binding of a regulatory protein specific to liver.

Glucokinase activity can be amplified or reduced in minutes by actions of the glucokinase regulatory protein (GKRP). The actions of this protein are influenced by small molecules such as glucose and fructose.

GKRP compete with glucose to bind with Glucokinase.

When glucose level is low: Glucokinase bound to GKRP (Nuclear localized and inactive)

When glucose level is high: GKRP not bound to Glucokinase (Active and cytoplasmic localization)

  • GKRP moves between nucleus and cytoplasm of the hepatocytes and may be tethered to the microfilament cytoskeleton.
  • It forms reversible 1:1 complexes with GK, and can move it from the cytoplasm into the nucleus. Hence glucokinase is segregated from the other enzymes of glycolysis in the cytosol.
  • GKRP acts as a competitive inhibitor with glucose, such that the enzyme activity is reduced to near-zero while bound.
  • GK: GKRP complexes are sequestered in the nucleus while glucose and fructose levels are low. Nuclear sequestration may serve to protect GK from degradation by cytoplasmic proteases.
Figure 2: Regulation of hexokinase IV (glucokinase) by sequestration in the nucleus.

Glucokinase exist in two conformations: 

1) Open conformation (Low activity): In the absence of glucose

2) Closed conformation: Glucokinase is active and no longer inhibited (In this conformation it is not accessible for glucokinase regulatory protein).

(The rise in cytosolic glucose concentration causes a concentration dependent transition of glucokinase open conformation to its close conformation)

What will happen to glucokinase during fasting?

During a fast, when blood glucose drops below 5 mM, fructose 6-phosphate binds to GKRP allowing it to bind tighter to glucokinase. This results in a strong inhibition of the enzyme. This mechanism ensures that the liver, at low blood glucose levels, does not compete with other organs, primarily the brain, for glucose. (The binding of GK with GKRP is much tighter in the presence of the allosteric effector fructose 6-phosphate. F6P enhances the ability of GKRP to bind and inactivate GK).

What will happen to glucokinase after a carbohydrate rich meal?

  • Immediately after a carbohydrate-rich meal, the glucose concentration in the cell rises.
  • It equilibrates with glucose in the nucleus by transport through the nuclear pores.
  • Glucose causes dissociation of the regulatory protein from glucokinase.
  • Glucokinase then enters the cytosol and begins to phosphorylate glucose.

3) Hexokinase IV is not inhibited by glucose 6- phosphate, and it can therefore continue to operate when the accumulation of glucose 6-phosphate completely inhibits hexokinases I–III.

Summary

When blood glucose levels are normal, glucose is phosphorylated mainly by hexokinases I-III, whereas when blood glucose levels are high glucose can be phosphorylated by glucokinase as well.

Difference between Hexokinase and glucokinase

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