Glycolysis 2 Regulation of glycolytic flux entry and exit of glycolytic metabolites and pyruvate metabolism Bioc 460 Spring 2008 Lecture 26 Miesfeld Glycolytic flux is regulated in part by PFK 1 activity a metabolic valve in the pathway Lactose intolerance is due to insufficient lactase enzyme This is the chemical structure of which glycolytic metabolite Key Concepts in Glycolysis Glucokinase hexokinase IV catalyzes reaction 1 in the glycolytic pathway in liver and pancreas cells when blood glucose levels are high Unlike hexokinase I glucokinase as a very low affinity for glucose and is not inhibited by glucose6P Therefore after a meal the liver accumulates glucose for glycogen synthesis and the insulin secretion pathway is activated in pancreatic cells Phosphofructokinase 1 PFK 1 is one of three metabolic valves in the glycolytic pathway PFK 1 is allosterically activated by fructose 2 6 BP AMP and ADP low energy charge whereas it is allosterically inhibited by ATP and citrate high energy charge AMP stabilizes the R form of the enzyme active and ATP stabilizes the T form inactive The activity of pyruvate kinase is controlled by both phosphorylation and allosteric regulation Disaccharide sugars maltose sucrose lactose are cleaved by specific enzymes to produce glucose and other monosaccharide sugars that enter the glycolytic pathway Glycolytic intermediates are metabolites in amino acid biosynthesis pentose phosphate pathway and triacylglyceride biosynthesis Regeneration of NAD in the cytosol is critical to maintaining glycolytic flux through the glyceraldehyde 3 P dehydrogenase reaction reaction 6 Glucokinase is a sensor of high glucose levels Hexokinase I high affinity for substrate Km for glucose is 0 1mM expressed in all tissues phosphorylates a variety of hexose sugars inhibited by the product of the reaction glucose 6 P Glucokinase Hexokinase IV low affinity for substrate Km for glucose is 10mM highly specific for glucose expressed primarily in liver and pancreatic cells not inhibited by glucose 6 P Two Major Roles of Glucokinase Role in liver cells When blood glucose levels are high both hexokinase I and glucokinase are active in liver cells whereas other tissues only have hexokinase 1 and their ability to take up glucose after a meal is unchanged Since phosphorylation traps glucose inside cells and reaction 1 of glycolysis same reaction catalyzed by both hexokinase 1 and glucokinase is highly favorable liver cells take up a disproportionate amount of the elevated blood glucose Role in pancreatic cells Glucokinase also sequesters glucose inside the pancreatic cells which initiates a complex signaling pathway leading to the release of insulin into the blood Since insulin signaling results in lowered blood glucose levels by activating glucose uptake in the muscle and fat cells adipose glucokinase is vital to glucose control Homeostatic blood glucose levels are 5mM which saturates hexokinase I activity in all tissues However after a carbohydrate rich meal glucose is plentiful blood glucose levels increase dramatically and the glucokinase reaction in liver and pancreas cells becomes a major contributor to the formation of glucose 6 P Since glucose 6 P is trapped in the cell because of the negative charge the liver and pancreas accumulate a large share of the blood glucose Glucokinase is a Sensor of Glucose Levels GLUT protein is a glucose transporter Glucose activation of glucokinase activity is at the level of protein synthesis i e elevated glucose in the cell leads to increased synthesis of glucokinase enzyme What happens to flux through the glycolytic pathway when glucokinase is activated by glucose Increased ATP levels stimulate membrane depolarization and subsequent calcuim uptake Making a glucokinase knock out mouse The importance of glucokinase in insulin secretion was confirmed using transgenic mice that lacked the glucokinase gene in pancreatic cells Since these mice cannot secrete insulin when blood glucose levels were high they developed insulin dependent diabetes Human metabolic diseases are often caused by non lethal recessive gene mutations in enzymes Over 150 mutations in the glucokinase gene have been found in humans with a form of diabetes called mature onset diabetes of the young MODY2 Allsoteric regulators control the activity of phosphofructokinase 1 and modulate glycolytic flux Phosophofructokinase 1 PFK 1 catalyzes reaction 3 in glycolysis to generate fructose 1 6 BP PFK 1 Phosphofructokinase 2 PFK 2 bifunctional enzyme that catalyzes the synthesis of fructose 2 6 BP F 2 6 BP a potent allosteric regulator of PFK 1 activity We will talk more about the synthesis of F 2 6 BP later in the course Allosteric regulation of PFK 1 PFK 1 exists as a homotetramer a dimer of dimers It is stable in two conformations the inactive T state or active R state similar to to the T and R conformations of hemoglobin The activity of PFK 1 is allosterically activated by binding of F 2 6 BP AMP and ADP and it is allosterically inhibited by citrate and ATP Allosteric regulators alter the activity of PFK 1 by binding to a region outside of the active site ATP and AMP binding to PFK 1 shifts the equilibrium between the T and R conformations What are the two roles of ATP in the PFK 1 catalyzed reaction The activity of liver pyruvate kinase is controlled by both phosphorylation and allosteric regulation When blood glucose levels are high glycolytic flux is stimulated in part by dephosphorylation of liver pyruvate kinase Conversely low blood glucose levels leads to phosphorylation and partial inactivation of pyruvate kinase Feed forward allosteric regulation by fructose1 6 BP and allosteric inhibition by ATP and alanine also control pyruvate kinase activity Based on what you know about the structure of pyruvate and alanine why do you think alanine is a negative regulator of pyruvate kinase activity Supply of Glycolytic Intermediates Disaccharide sugars are common nutrients in our diet and provide much of the carbohydrate used for energy conversion Maltose is from starch Sucrose is table sugar Lactose is from milk Glycerol is a glycolytic intermediate derived from the degradation of triacylglycerides Lactose intolerance occurs in most adults as a result of decreased lactase enzyme production High levels of intestinal lactose causes osmosis of water into the intestine leading to diarrhea and moreover anaerobic intestinal bacteria metabolize the extra lactose to produce H2 and CH4 gas
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