|Topic:||Glycolysis Reactions .|
|Details:||The pathway of glycolysis can be seen as consisting of two separate phases. The first is the chemical priming phase requiring energy in the form of ATP, and the second is considered the energy-yielding phase. In the first phase, two equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP). In the second phase F1,6BP is degraded to pyruvate, with the production of four equivalents of ATP and two equivalents of NADH.
The Hexokinase Reaction:
The ATP-dependent phosphorylation of glucose to form glucose 6-phosphate (G6P) is the first reaction of glycolysis, and is catalyzed by tissue-specific isozymes known as hexokinases. The phosphorylation accomplishes two goals: First, the hexokinase reaction converts non-ionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars. Second, the otherwise biologically inert glucose becomes activated into a labile form capable of being further metabolized.
Four mammalian isozymes of ATP-dependent hexokinase are known (Types I–IV: HK1, HK2, HK3, and HK4), with the HK4 isoform more commonly referred to as glucokinase and its gene designated as GCK. All four mammalian hexokinases/glucokinase function as monomeric enzymes. Hexokinases 1, 2, and 3 can all phosphorylate other hexose sugars (at their normal physiological concentrations) in addition to glucose, whereas glucokinase (HK4) is only physiologically active towards glucose. The HK1 gene is located on chromosome 10q22.1 that spans 75 kb and is composed of 28 exons that generate ten alternatively spliced mRNAs that encode ubiquitously expressed and tissue-specific isoforms of the enzyme. The HK1 mRNA encodes the ubiquitously expressed isoform that is a 917 amino acid enzyme. The HK1-R isoform is the erythroid-specific isoform and is a 916 amino acid enzyme. There are four mRNAs that express the HK1-ta/tb isoform which is 921 amino acid testes-specific isoform. The HK1-td isoform is also a testes-specific isoform of 905 amino acids. The other three mRNAs encode HK1 isoform a (952 amino acids), isoform b (889 animo acids), and isoform c (885 amino acids). The HK2 gene is located on chromosome 2p12 that spans 50 kb and is composed of 18 exons that encode a 917 amino acid protein. The HK3 gene is located on chromosome 5q35.2 and is composed of 22 exons that encode a protein of 923 amino acids. The GCK (HK4) gene is located on chromosome 7p13 and is composed of 13 exons. As a result of alternative promoter useage and alternative splicing the GCK gene gives rise to seven different mRNAs and seven different protein isoforms. GCK isoform 1 is a pancreas specific enzyme of 465 amino acids. GCK isoform 2 is one of two liver-specific enzymes with this protein being the predominant form found in the liver. GCK isoform 2 is a 466 amino acid protein and isoform 3 is a 464 amin acid protein. The other four GCK mRNAs (encoding isoforms 4, 5, 6, and 7) have, as yet, not been assoicated with tissue-specific patterns of expression.
As indicated, the HK1 isoform is ubiquitously expressed in most mammalian tissues. HK2 expression is normally restricted to insulin-sensitive tissues such as adipose tissue, skeletal and cardiac muscle. However, high level HK2 expression is observed in cancer cells and this switch is associated with poor survival rates. Activated expression of HK2 in cancer cells is associated with a loss in expression of the tumor suppressor, p53. HK3 is normally expressed at low levels. As indicated, glucokinase (HK4) expression is essentially restricted to hepatocytes and pancreatic β-cells with low expression seen within the hypothalamus. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate, i.e. only in the postprandial state.
Sigmoidal kinetics has been observed for other monomeric enzymes in cases of random addition of substrates, but this is not the case for glucokinase. The catalytic cycle for glucokinase involves an ordered addition of substrates where glucose is known to bind first followed by ATP binding. The mechanism of positive cooperativity observed for monomeric enzymes was indeed first proposed for glucokinase and is called the mnemonic model. The mnemonic mechanism of cooperativity for glucokinase involves an equilibrium between two conformational states of the enzyme that exhibit vastly different glucose affinities. Within cells there is a large predominance of the low affinity conformation of glucokinase in the absence of glucose. As glucose concentrations rise, there is a slow interconversion between the conformational states with conversion from the low affinity to the high affinity state strongly accelerated upon glucose binding to the active site of the enzyme. Only the high affinity form of glucokinase is catalytically competent, and the rate of glucose phosphorylation is very fast compared to the rate of glucose-induced conformational change. In addition to the unique kinetic parameters of glucokinase, compared to those of HK1, HK2, and HK3, glucokinase is also regulated through interaction with a regulatory protein (see the Regulation of Glycolysis section below), whereas the other three enzymes are not.
The high Km, for glucose, of hepatic glucokinase allows the liver to buffer blood glucose. After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes the liver preferentially to trap and to store circulating glucose. When blood glucose falls to very low levels, tissues such as liver and kidney, which contain glucokinases but are not highly dependent on glucose, do not continue to use the meager glucose supplies that remain available. At the same time, tissues such as the brain, which are critically dependent on glucose, continue to scavenge blood glucose using their low Km hexokinases, and as a consequence their viability is protected. Under various conditions of glucose deficiency, such as long periods between meals, the liver is stimulated to supply the blood with glucose through the pathway of gluconeogenesis. The levels of glucose produced during gluconeogenesis are insufficient to activate glucokinase, allowing the glucose to pass out of hepatocytes and into the blood.
The regulation of hexokinase and glucokinase activities is also different. Hexokinases 1, 2, and 3 are feed-back inhibited by physiological accumulation of the product (G6P) of their reactions, whereas glucokinase is not inhibited by physiological levels of G6P. The relative lack of product inhibition of glucokinase further insures liver accumulation of glucose stores during times of glucose excess, while favoring peripheral glucose utilization when glucose is required to supply energy to peripheral tissues. The activity of the hexokinases is also regulated by inorganic phosphate (Pi). HK2 and HK3 are further inhibited by Pi, whereas the G6P inhibition of HK1 is antagonized by low concentrations of Pi while high Pi concentrations contribute to further G6P inhibition of HK1. The subcellular localization is also distinct for various hexokinase isoforms with HK1 being associated with the mitochondria while GCK moves between the nucleus and the cytosol. The HK2 enzyme can also associate with the mitochondria but it is not known if this is a physiologically relevant interaction as it is for HK1. The interaction of HK1 with the actively phosphorylating mitochondria, and its selective use of intramitochondrial ATP as a substrate is thought to facilitate coordination of glycolysis with the terminal oxidative stages of glucose metabolism which occurs within the mitochondria. This would ensure that the rate of overall glucose oxidation is commensurate with cellular energy demands while also avoiding excessive production of lactate.
The primary mechanism of glucokinase regulation is its sequestration to the nucleus by the protein, glucokinase regulatory protein, GKRP (see the Regulation of Glycolysis section below for details). Although not physiologically inhibited by its product, hepatic glucokinase is inhibited by long-chain fatty acids (LCFA). In contrast, LCFAs do not inhibit the other forms of hexokinase. The ability of LCFAs to inhibit hepatic glucokinase is one of the mechanisms by which fatty acids inhibit glucose uptake into the liver. The inhibition of hepatic glucose uptake by LCFA is responsible, in part, for the hyperglycemia observed in obesity.
Hypothalamic expression of the GCK gene plays an important role in the regulation of dietary glucose intake in particular, and overall feeding behavior in general. The primary hypothalamic cells expressing glucokinase are within the arcuate nucleus, ARC. Expression of the hypothalamic GCK gene increases specifically within the ARC in response to fasting. Manipulation of GCK expression within the ARC of experimental animals alters glucose intake. Increased GCK expression in the ARC results in increased glucose ingestion, whereas, decreased GCK expression results in reduced glucose ingestion. These observations indicate that ARC expression of GCK underlies the phenomenon of carbohydrate craving.
In addition to the ATP-dependent glucose phosphorylating hexokinases/glucokinase, an additional glucose phosphorylating enzyme was identified in 2004. This enzyme is dependent upon ADP for activity and not ATP. This ADP-dependent glucokinase (ADP-GK) is encoded by the ADPGK gene which is located on chromosome 15q24.1 and is composed of 9 exons that encode a 496 amino acid precursor protein. Expression of the ADPGK gene is seen in numerous tissues implying that it serves a housekeeping role with respect to glucose metabolism. The ADP-GK enzyme is highly specific for glucose with a Km for this substrate of around 0.1 mM. ADP-GK is inhibited by both high concentrations of glucose and by AMP. It is believed that this glucose phosphorylating enzyme is physiologically important during periods of ischemia/hypoxia.
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