Bioc 460 - Dr. Miesfeld Spring 2008 1 of 9 pages Figure 1. Glycolysis 2 Supplemental reading Key Concepts - Regulation of the Glycolytic Pathway • Glucokinase is a molecular sensor of high glucose levels • Allosteric control of phosphofructokinase activity • Supply and demand of glycolytic intermediates - Metabolic Fate of Pyruvate KEY CONCEPT QUESTIONS IN GLYCOLYSIS: How do substrate availability and enzyme activity levels control glycolytic flux? Why is muscle lactate dehydrogenase activity required for short bursts of intense exercise? Biochemical Applications of Glycolysis: Inherited genetic defects in metabolic enzymes can give rise to human diseases that are caused by decreased levels of enzyme or altered enzyme activities (complete loss of the enzyme is often embryonic lethal). Enzyme deficiencies in gluckokinase, lactate dehydrogenase, and fructose-1P aldolase, all lead to metabolic diseases. REGULATION OF THE GLYCOLYTIC PATHWAY Irreversible reactions in metabolic pathways are called rate-limiting steps because the level of enzyme activity can be low even when substrate levels are high. Rate-limiting enzymes in metabolic pathways serve as regulated “valves” that are opened or closed in response to cellular conditions. As illustrated in figure 1, reversible steps in glycolysis and gluconeogenesis operate in both pathways, whereas, irreversible steps have actual changes in free energies (ΔG) that are highly negative and require pathway-specific enzymes. • Glucokinase is a molecular sensor of high glucose levels Four hexokinase genes have been identified in humans (hexokinase I-IV), all of which are capable of converting glucose to glucose-6-P at the expense of ATP hydrolysis (step 1 of glycolysis). We have already described one of these, hexokinase I (reaction 1), which has a high affinity for substrate (Km for glucose isBioc 460 - Dr. Miesfeld Spring 2008 2 of 9 pages Figure 3. ~0.1mM), is expressed in all tissues, phosphorylates a variety of hexose sugars, and is inhibited by the product of the reaction, glucose-6-P. In contrast, hexokinase IV, also known as glucokinase, has a low affinity for substrate (Km for glucose is ~10mM), is highly specific for glucose, is expressed primarily in liver and pancreatic cells and is not inhibited by glucose-6-P. This difference in tissue expression and glucose affinity between hexokinase and glucokinase plays an important role in controlling blood glucose levels, which ultimately controls rates of glycolytic flux in all cells by limiting substrate availability. As suggested by the different Km values of hexokinase and glucokinase for glucose, substrate saturation curves for these two enzymes look markedly different as shown in figure 2. Since blood glucose levels are maintained around 5mM, significant levels of glucose phosphorylation by glucokinase only occur under conditions of high glucose, such as after consuming a carbohydrate-rich meal. Moreover, since glucokinase is not inhibited by glucose-6-P, it is able to continue functioning even if flux through glycolysis cannot keep up with product formation. The role of glucokinase in liver cells is to trap the extra glucose that is available from the diet so that it can be stored as glycogen for an energy source later. Figure 2. By being active in liver cells only when glucose concentrations exceed normal limits (>5mM), glucokinase ensures that the liver is the major sink for dietary glucose, and at the same time, is able to efficiently remove glucose from the blood to help restore normal blood glucose concentrations. Another important function of glucokinase is to act as a glucose sensor in pancreatic β cells where glucokinase enzyme levels are activated by increased glucose import mediated by glucose transporter proteins (GLUT proteins). As shown in figure 3, when the concentration of glucose in the blood is elevated, glucose import intoBioc 460 - Dr. Miesfeld Spring 2008 3 of 9 pages pancreatic β cells leads to higher glucokinase enzyme levels resulting in increased flux through glycolysis and net ATP synthesis. This increase in ATP levels causes inhibition of ATP-sensitive K+ channels, membrane depolarization, and activation of voltage-gated Ca2+ channels. Elevated levels of intracellular Ca2+ triggers fusion of insulin-containing vesicles with the plasma membrane and subsequent release of insulin into the blood. This intracellular signaling pathway links glucose uptake in pancreatic β cells with insulin release. The importance of pancreatic glucokinase in insulin secretion was confirmed using transgenic mice in which the glucokinase gene was specifically deleted in pancreatic β cells. These glucokinase-deficient mice were defected in glucose-stimulated insulin secretion and became hyperglycemic, eventually developing diabetic symptoms due to chronic elevated blood glucose. Well over 100 mutations in the human glucokinase gene have been identified and it is now known that a form of type II diabetes in humans, called maturity-onset diabetes of the young (MODY2), is caused by defects in pancreatic glucokinase activity. Allosteric control of phosphofructokinase activity Of all the enzymes in glycolysis, phosphofructokinase is the best characterized because of its vital role in controlling flux through the pathway. There are actually two phosphofructokinase isozymes (distinct genes that encode proteins with similar functions), phosophofructokinase-1 (PFK-1) which catalyzes reaction 3 in glycolysis, and phosphofructokinase-2 (PFK-2) a bifunctional enzyme that catalyzes the synthesis of fructose-2,6-bisophosphate (F-2,6-BP), a potent allosteric regulator of PFK-1 activity (discussed in lecture 35). The PFK-1 reaction in glycolysis is irreversible and functions as one of three metabolic “valves” that controls flux through the pathway (the other two are the hexokinase and pyruvate kinase reactions). Figure 4 illustrates that AMP, ADP and F-2,6-BP are activators of PFK-1 activity, and ATP and citrate function as inhibitors. Figure 4. PFK-1 is an allosteric enzyme that exists as a tetramer (a dimer of dimers) in either of two conformations, the inactive T state or active R state, analogous to the hemoglobin tetramer. The equilibrium between T and R states in a cell is controlled by allosteric effector molecules which bind to a regulatory site outside of the substrate binding pocket. ATP and citrate are negative effector
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