Glucokinase


Glucokinase is an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate. Glucokinase is expressed in cells of the liver and pancreas of humans and most other vertebrates. In each of these organs it plays an important role in the regulation of carbohydrate metabolism by acting as a glucose sensor, triggering shifts in metabolism or cell function in response to rising or falling levels of glucose, such as occur after a meal or when fasting. Mutations of the gene for this enzyme can cause unusual forms of diabetes or hypoglycemia.
Glucokinase is a hexokinase isozyme, related homologously to at least three other hexokinases. All of the hexokinases can mediate phosphorylation of glucose to glucose-6-phosphate, which is the first step of both glycogen synthesis and glycolysis. However, glucokinase is coded by a separate gene and its distinctive kinetic properties allow it to serve a different set of functions. Glucokinase has a lower affinity for glucose than the other hexokinases do, and its activity is localized to a few cell types, leaving the other three hexokinases as more important preparers of glucose for glycolysis and glycogen synthesis for most tissues and organs. Because of this reduced affinity, the activity of glucokinase, under usual physiological conditions, varies substantially according to the concentration of glucose. Additionally, unlike other hexokinase isozymes, glucokinase is not subject to feedback inhibition by physiological levels its product, glucose-6-phosphate, allowing for continuing function even under high product production.

Nomenclature

Alternative names for this enzyme are: human hexokinase IV, hexokinase D, and ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1. The common name, glucokinase, is derived from its relative specificity for glucose under physiologic conditions.
Some biochemists have argued that the name glucokinase should be abandoned as misleading, as this enzyme can phosphorylate other hexoses in the right conditions, and there are distantly related enzymes in bacteria with more absolute specificity for glucose that better deserve the name and the EC . Nevertheless, glucokinase remains the name preferred in the contexts of medicine and mammalian physiology.
Another mammalian glucose kinase, ADP-specific glucokinase, was discovered in 2004. The gene is distinct and similar to that of primitive organisms. It is dependent on ADP rather than ATP, and the metabolic role and importance remain to be elucidated.

Catalysis

Substrates and products

The principal substrate of physiological importance of glucokinase is glucose, and the most important product is glucose-6-phosphate. The other necessary substrate, from which the phosphate is derived, is adenosine triphosphate, which is converted to adenosine diphosphate when the phosphate is removed. The reaction catalyzed by glucokinase is shown in the inset.
ATP participates in the reaction in a form complexed to magnesium as a cofactor. Furthermore, under certain conditions, glucokinase, like other hexokinases, can induce phosphorylation of other hexoses and similar molecules. Therefore, the general glucokinase reaction is more accurately described as:
Among the hexose substrates are mannose, fructose, and glucosamine, but the affinity of glucokinase for these requires concentrations not found in cells for significant activity. Nonetheless, the specificity for glucose is much less clear than was long thought, and by the usual criteria for specificity fructose is a good substrate.

Kinetics

Three important kinetic properties distinguish glucokinase from the other hexokinases, allowing it to function in a special role as glucose sensor.
  1. Glucokinase has a lower affinity for glucose than the other hexokinases. Glucokinase changes conformation and/or function in parallel with rising glucose concentrations in the physiologically important range of 4–10 M. It is half-saturated at a glucose concentration of about 8 mM.
  2. Glucokinase is not inhibited by physiological concentrations of its product, glucose-6-phosphate. This allows continued signal output amid significant amounts of its product.
  3. Another distinctive property of glucokinase is its moderate cooperativity with glucose, with a Hill coefficient of about 1.7.
These features allow it to regulate a "supply-driven" metabolic pathway. That is, the rate of reaction is driven by the supply of glucose, not by the demand for end products.
Because of the cooperativity, the kinetic interaction of glucokinase with glucose does not follow classical Michaelis-Menten kinetics. Rather than a Km for glucose, it is more accurate to describe a half-saturation level S0.5, the concentration at which the enzyme is 50% saturated and active.
The S0.5 and h result in an inflection of the curve enzyme activity as a function of glucose concentration at about 4 mM. In other words, at a glucose concentration of about 72 mg/dL, which is near the low end of the normal range, glucokinase activity is most sensitive to small changes in glucose concentration.
As glucokinase is a monomeric enzyme with only a single binding site for glucose the cooperativity cannot be explained in terms of classical models of equilibrium cooperativity, but requires a kinetic explanation, such as a slow-transition model or a "memonical" model that invokes enzyme memory.
The kinetic relationship with the other substrate, MgATP, can be described by classical Michaelis-Menten kinetics, with an affinity at about 0.3–0.4 mM, well below a typical intracellular concentration of 2.5 mM. The fact that there is nearly always an excess of ATP available implies that ATP concentration rarely influences glucokinase activity.
The maximum specific activity of glucokinase when saturated with both substrates is 62/s.
The pH optimum of human glucokinase was identified only recently and is surprisingly high, at pH 8.5–8.7.
A "minimal mathematical model" has been devised based on the above kinetic information to predict the beta cell glucose phosphorylation rate of normal glucokinase and the known mutations. The BGPR for wild type glucokinase is about 28% at a glucose concentration of 5 mM, indicating that the enzyme is running at 28% of capacity at the usual threshold glucose for triggering insulin release.

Mechanism

The sulfhydryl groups of several cysteines surround the glucose binding site. All except cys 230 are essential for the catalytic process, forming multiple disulfide bridges during interaction with the substrates and regulators. At least in the beta cells, the ratio of active to inactive glucokinase molecules is at least partly determined by the balance of oxidation of sulfhydryl groups or reduction of disulfide bridges.
These sulfhydryl groups are quite sensitive to the oxidation status of the cells, making glucokinase one of the components most vulnerable to oxidative stress, especially in the beta cells.

Interactive pathway map

Structure

Glucokinase is a monomeric protein of 465 amino acids and a molecular weight of about 50 kDa. There are at least two clefts, one for the active site, binding glucose and MgATP, and the other for a putative allosteric activator that has not yet been identified.
This is about half the size of the other mammalian hexokinases, which retain a degree of dimeric structure. Several sequences and the three-dimensional structure of the key active sites are highly conserved both in intra-species homologs and across species from mammals to yeast. The ATP binding domain, for example, are shared with hexokinases, bacterial glucokinases, and other proteins, and the common structure is termed an actin fold.

Genetics

Human glucokinase is coded for by the GCK gene on chromosome 7. This single autosomal gene has 10 exons.
Genes for glucokinase in other animals are homologous to human GCK.
A distinctive feature of the gene is that it begins with two promoter regions. The first exon from the 5' end contains two tissue-specific promoter regions. Transcription can begin at either promoter so that the same gene can produce a slightly different molecule in liver and in other tissues. The two isoforms of glucokinase differ only by 13–15 amino acids at the N-terminal end of the molecule, which produces only a minimal difference in structure. The two isoforms have the same kinetic and functional characteristics.
The first promoter from the 5' end, referred to as the "upstream" or neuroendocrine promoter, is active in pancreatic islet cells, neural tissue, and enterocytes to produce the "neuroendocrine isoform" of glucokinase. The second promoter, the "downstream" or liver promoter, is active in hepatocytes and directs production of the "liver isoform." The two promoters have little or no sequence homology and are separated by a 30 kbp sequence which has not yet been shown to incur any functional differences between isoforms. The two promoters are functionally exclusive and governed by distinct sets of regulatory factors, so that glucokinase expression can be regulated separately in different tissue types. The two promoters correspond to two broad categories of glucokinase function: In liver, glucokinase acts as the gateway for the "bulk processing" of available glucose, while, in the neuroendocrine cells, it acts as a sensor, triggering cell responses that affect body-wide carbohydrate metabolism.

Distribution among organ systems

Glucokinase has been discovered in specific cells in four types of mammalian tissue: liver, pancreas, small intestine, and brain. All play crucial roles in responding to rising or falling levels of blood glucose.
  • The predominant cells of the liver are the hepatocytes, and GK is found exclusively in these cells. During digestion of a carbohydrate meal, when blood glucose is plentiful and insulin levels are high, hepatocytes remove glucose from the blood and store it as glycogen. After completion of digestion and absorption, the liver manufactures glucose from both non-glucose substrates and glycogen, and exports it into the blood, to maintain adequate blood glucose levels during fasting. Because GK activity rises rapidly as the glucose concentration rises, it serves as a central metabolic switch to shift hepatic carbohydrate metabolism between fed and fasting states. Phosphorylation of glucose to glucose-6-phosphate by GK facilitates storage of glucose as glycogen and disposal by glycolysis. The separate liver promoter allows glucokinase to be regulated differently in hepatocytes than in the neuroendocrine cells.
  • Neuroendocrine cells of the pancreas, gut, and brain share some common aspects of glucokinase production, regulation, and function. These tissues are collectively referred to as "neuroendocrine" cells in this context.
  • *Beta cells and alpha cells of the pancreatic islets
  • **Beta cells release insulin in response to rising levels of glucose. Insulin enables many types of cells to import and use glucose, and signals the liver to synthesize glycogen. Alpha cells produce less glucagon in response to rising glucose levels, and more glucagon if blood glucose is low. Glucagon serves as a signal to the liver to break down glycogen and release glucose into the blood. Glucokinase in beta cells serves as a glucose sensor, amplifying insulin secretion as blood glucose rises.
  • **In the pancreatic beta-cell, glucokinase is a key regulator enzyme. Glucokinase is very important in the regulation of insulin secretion and has been known as the pancreatic beta-cell sensor. Mutations in the gene encoding glucokinase can cause both hyperglycemia and hypoglycemia because of its central role in the regulation of insulin release.
  • *Glucose-sensitive neurons of the hypothalamus
  • **In response to rising or falling levels of glucose, cells in the hypothalamus polarize or depolarize. Among the neuroendocrine reactions of the central nervous system to hypoglycemia is activation of the adrenergic responses of the autonomic nervous system. Glucokinase likely serves as a glucose signal here as well. Glucokinase has also been found in cells of the anterior pituitary.
  • *Enterocytes of the small intestine
  • **This is the least-understood of the glucokinase sensor systems. It seems likely that responses to incoming glucose during digestion play a role in the incretin amplification of insulin secretion during a meal, or in the generation of satiety signals from gut to brain.