Hexokinase

A hexokinase is an enzyme 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 can transfer an inorganic phosphate group from ATP to a substrate.

Hexokinases should not be confused with glucokinase, which is a specific isoform of hexokinase. While other hexokinases are capable of phosphorylating several hexoses, glucokinase acts with a 50-fold lower substrate affinity and its only hexose substrate is glucose.

Variation

Genes that encode hexokinase have been discovered in every domain of life, and exist among a variety of species that range from bacteria, yeast, and plants to humans and other vertebrates. They are categorized as actin fold proteins, sharing a common ATP binding site core that is surrounded by more variable sequences which determine substrate affinities and other properties.

Several hexokinase isoforms or isozymes that provide different functions can occur in a single species.

Reaction

The intracellular reactions mediated by hexokinases can be typified as:

Hexose-CH₂OH + MgATP2−
→ Hexose-CH₂O-PO2−
3 + MgADP−
+ H⁺

where hexose-CH₂OH represents any of several hexoses (like glucose) that contain an accessible -CH₂OH moiety.

Consequences of hexose phosphorylation

Phosphorylation of a hexose such as glucose often limits it to a number of intracellular metabolic processes, such as glycolysis or glycogen synthesis. This is because phosphorylated hexoses are charged, and thus more difficult to transport out of a cell.

In patients with essential fructosuria, metabolism of fructose by hexokinase to fructose-6-phosphate is the primary method of metabolizing dietary fructose; this pathway is not significant in normal individuals.

Size of different isoforms

Most bacterial hexokinases are approximately 50 kD in size. Multicellular organisms such as plants and animals often have more than one hexokinase isoform. Most are about 100 kD in size and consist of two halves (N and C terminal), which share much sequence homology. This suggests an evolutionary origin by duplication and fusion of a 50kD ancestral hexokinase similar to those of bacteria.

Types of mammalian hexokinase

There are four important mammalian hexokinase isozymes (EC 2.7.1.1) that vary in subcellular locations and kinetics with respect to different substrates and conditions, and physiological function. They are designated hexokinases I, II, III, and IV or hexokinases A, B, C, and D.

Hexokinases I, II, and III

Hexokinases I, II, and III are referred to as "low-K_m" isozymes because of a high affinity for glucose (below 1 mM). Hexokinases I and II follow Michaelis-Menten kinetics at physiologic concentrations of substrates. All three are strongly inhibited by their product, glucose-6-phosphate. Molecular weights are around 100 kD. Each consists of two similar 50kD halves, but only in hexokinase II do both halves have functional active sites.

Hexokinase I/A is found in all mammalian tissues, and is considered a "housekeeping enzyme," unaffected by most physiological, hormonal, and metabolic changes.
Hexokinase II/B constitutes the principal regulated isoform in many cell types and is increased in many cancers. It is the hexokinase found in muscle and heart. Hexokinase II is also located at the mitochondria outer membrane so it can have direct access to ATP.^[2]
Hexokinase III/C is substrate-inhibited by glucose at physiologic concentrations. Little is known about the regulatory characteristics of this isoform.

Hexokinase IV ("glucokinase")

Mammalian hexokinase IV, also referred to as glucokinase, differs from other hexokinases in kinetics and functions.

The location of the phosphorylation on a subcellular level occurs when glucokinase translocates between the cytoplasm and nucleus of liver cells. Glucokinase can only phosphorylate glucose if the concentration of this substrate is high enough; its Km for glucose is 100 times higher than that of hexokinases I, II, and III.

Hexokinase IV is monomeric, about 50kD, displays positive cooperativity with glucose, and is not allosterically inhibited by its product, glucose-6-phosphate.

Hexokinase IV is present in the liver, pancreas, hypothalamus, small intestine, and perhaps certain other neuroendocrine cells, and plays an important regulatory role in carbohydrate metabolism. In the beta cells of the pancreatic islets, it serves as a glucose sensor to control insulin release, and similarly controls glucagon release in the alpha cells. In hepatocytes of the liver, glucokinase responds to changes of ambient glucose levels by increasing or reducing glycogen synthesis.

Hexokinase in glycolysis

Glucose is unique in that it can be used to produce ATP by all cells in both the presence and absence of molecular oxygen (O₂). The first step in glycolysis is the phosphorylation of glucose by hexokinase.

D-Glucose	Hexokinase		α-D-Glucose-6-phosphate

	ATP	ADP

Compound C00031 at KEGG Pathway Database. Enzyme 2.7.1.1 at KEGG Pathway Database. Compound C00668 at KEGG Pathway Database. Reaction R01786 at KEGG Pathway Database.

By catalyzing the phosphorylation of glucose to yield glucose 6-phosphate, hexokinases maintain the downhill concentration gradient that favors the facilitated transport of glucose into cells. This reaction also initiates all physiologically relevant pathways of glucose utilization, including glycolysis and the pentose phosphate pathway.^[3] The addition of a charged phosphate group at the 6-position of hexoses also ensures 'trapping' of glucose and 2-deoxyhexose glucose analogs (e.g. 2-deoxyglucose, and 2-fluoro-2-deoxyglucose) within cells, as charged hexose phosphates cannot easily cross the cell membrane.

Association with mitochondria

Hexokinases I and II can associate physically to the outer surface of the external membrane of mitochondria through specific binding to a porin, or voltage dependent anion channel. This association confers hexokinase direct access to ATP generated by mitochondria, which is one of the two substrates of hexokinase. Mitochondrial hexokinase is highly elevated in rapidly growing malignant tumor cells, with levels up to 200 times higher than normal tissues. Mitochondrially bound hexokinase has been demonstrated to be the driving force^[4] for the extremely high glycolytic rates that take place aerobically in tumor cells (the so-called Warburg effect described by Otto Heinrich Warburg in 1930).

Hydropathy plot

Hydropathy plot of hexokinase

The potential transmembrane portions of a protein can be detected by hydropathy analysis. A hydropathy analysis uses an algorithm that quantifies the hydrophobic character at each position along the polypeptide chain. One of the accepted hydropathy scales is that of Kyte and Doolittle which relies on the generation of hydropathy plots. In these plots, the negative numbers represent hydrophilic regions and the positive numbers represent hydrophobic regions on the y-axis. A potential transmembrane domain is about 20 amino acids long on the x-axis.

A hydropathy analysis of hexokinase in yeast has been created by these standards. It appears as if hexokinase possesses a single potential transmembrane domain located around amino acid 400. Therefore, hexokinase is most likely not an integral membrane protein in yeast.^[5]

References

↑ PDB: 3O08; Kuettner EB, Kettner K, Keim A, Svergun DI, Volke D (2010). "Crystal structure of dimeric KlHxk1 in crystal form I". doi:10.2210/pdb3o08/pdb.
↑ uniprot.org http://www.uniprot.org/uniprot/P52789#subcellular_location. Missing or empty |title= (help)
↑ Robey, RB; Hay, N (2006). "Mitochondrial hexokinases, novel mediators of the antiapoptotic effects of growth factors and Akt". Oncogene. 25 (34): 4683–96. doi:10.1038/sj.onc.1209595. PMID 16892082.
↑ Bustamante E, Pedersen P (1977). "High aerobic glycolysis of rat hepatoma cells in culture: role of mitochondrial hexokinase". Proc Natl Acad Sci USA. 74 (9): 3735–9. Bibcode:1977PNAS...74.3735B. doi:10.1073/pnas.74.9.3735. PMC 431708. PMID 198801.
↑ Bowen, R. A. Molecular Toolkit: Protein Hydrophobicity Plots. Colorado State University, 1998. Web. 15 Nov. 2010. <http://www.vivo.colostate.edu/molkit/index.html>

Glycolysis metabolic pathway

Glucose	Hexokinase		Glucose 6-phosphate	Glucose-6-phosphate isomerase	Fructose 6-phosphate	phosphofructokinase-1		Fructose 1,6-bisphosphate	Fructose-bisphosphate aldolase
	ATP	ADP				ATP	ADP

Dihydroxyacetone phosphate		Glyceraldehyde 3-phosphate	Triosephosphate isomerase		Glyceraldehyde 3-phosphate	Glyceraldehyde-3-phosphate dehydrogenase			1,3-Bisphosphoglycerate
						NAD⁺ + P_i	NADH + H⁺
	+			2				2

Phosphoglycerate kinase

3-Phosphoglycerate

Phosphoglycerate mutase

2-Phosphoglycerate

Phosphopyruvate hydratase(Enolase)

Phosphoenolpyruvate

Pyruvate kinase

Pyruvate

ADP

ATP

H₂O

ADP

ATP

Metabolism: carbohydrate metabolism: glycolysis/gluconeogenesis enzymes

Glycolysis

Gluconeogenesis only

to oxaloacetate:	Pyruvate carboxylase Phosphoenolpyruvate carboxykinase

from lactate (Cori cycle):	Lactate dehydrogenase

from alanine (Alanine cycle):	Alanine transaminase

from glycerol:	Glycerol kinase Glycerol dehydrogenase

Regulatory

Transferases: phosphorus-containing groups (EC 2.7)

2.7.1-2.7.4:
phosphotransferase/kinase
(PO₄)

2.7.1: OH acceptor	Hexo- Gluco- Fructo- Hepatic Galacto- Phosphofructo- 1 Liver Muscle Platelet 2 Riboflavin Shikimate Thymidine ADP-thymidine NAD⁺ Glycerol Pantothenate Mevalonate Pyruvate Deoxycytidine PFP Diacylglycerol Phosphoinositide 3 Class I PI 3 Class II PI 3 Sphingosine Glucose-1,6-bisphosphate synthase

2.7.2: COOH acceptor	Phosphoglycerate Aspartate kinase

2.7.3: N acceptor	Creatine

2.7.4: PO₄ acceptor	Phosphomevalonate Adenylate Nucleoside-diphosphate Uridylate Guanylate Thiamine-diphosphate

2.7.6: diphosphotransferase
(P₂O₇)

2.7.7: nucleotidyltransferase
(PO₄-nucleoside)

Polymerase

DNA polymerase	DNA-directed DNA polymerase I II III IV V RNA-directed DNA polymerase Reverse transcriptase Telomerase DNA nucleotidylexotransferase/Terminal deoxynucleotidyl transferase

RNA nucleotidyltransferase	RNA polymerase/DNA-directed RNA polymerase RNA polymerase I RNA polymerase II RNA polymerase III RNA polymerase IV Primase RNA-dependent RNA polymerase PNPase

Phosphorolytic
3' to 5' exoribonuclease

Nucleotidyltransferase

Guanylyltransferase

mRNA capping enzyme

Other

2.7.8: miscellaneous

Phosphatidyltransferases	CDP-diacylglycerol—glycerol-3-phosphate 3-phosphatidyltransferase CDP-diacylglycerol—serine O-phosphatidyltransferase CDP-diacylglycerol—inositol 3-phosphatidyltransferase CDP-diacylglycerol—choline O-phosphatidyltransferase

Glycosyl-1-phosphotransferase	N-acetylglucosamine-1-phosphate transferase

2.7.10-2.7.13: protein kinase
(PO₄; protein acceptor)

2.7.10: protein-tyrosine	see tyrosine kinases

2.7.11: protein-serine/threonine	see serine/threonine-specific protein kinases

2.7.12: protein-dual-specificity	see serine/threonine-specific protein kinases

2.7.13: protein-histidine	Protein-histidine pros-kinase Protein-histidine tele-kinase Histidine kinase

Enzymes

Activity	Active site Binding site Catalytic triad Oxyanion hole Enzyme promiscuity Catalytically perfect enzyme Coenzyme Cofactor Enzyme catalysis Enzyme kinetics Lineweaver–Burk plot Michaelis–Menten kinetics

Regulation	Allosteric regulation Cooperativity Enzyme inhibitor

Classification	EC number Enzyme superfamily Enzyme family List of enzymes

Types	EC1 Oxidoreductases (list) EC2 Transferases (list) EC3 Hydrolases (list) EC4 Lyases (list) EC5 Isomerases (list) EC6 Ligases (list)

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