© 2012 Pearson Education, Inc. CHEMOTROPIC ENERGY:GLYCOLYSIS AND FERMENTATION Chapter 9

© 2012 Pearson Education, Inc. Glycolysis Generates ATP by Catabolizing Glucose to Pyruvate Glycolysis (or the glycolytic pathway) is a ten- step reaction sequence that converts one glucose molecule into two molecules of pyruvate Pyruvate is a three-carbon compound Both ATP and NADH are produced

© 2012 Pearson Education, Inc. Figure 9-6

© 2012 Pearson Education, Inc. Figure 9-7

© 2012 Pearson Education, Inc. Glycolysis is present in all organisms Glycolysis is common to both aerobic and anaerobic organisms In most cells the enzymes for glycolysis are found in the cytosol

© 2012 Pearson Education, Inc. Glycolysis in Overview In the absence of oxygen glycolysis leads to fermentation In the presence of oxygen glycolysis leads to aerobic respiration

© 2012 Pearson Education, Inc. Important features of the glycolytic pathway are The initial input of ATP (Gly-1) The sugar splitting reaction in which glucose is split into two three-carbon molecules The oxidative event that generates NADH (Gly-6) The two steps at which the reaction sequence is coupled to ATP generation (Gly-7 and Gly-10)

© 2012 Pearson Education, Inc. The glycolytic pathway can be divided into three phases Phase I: the preparatory and cleavage steps Phase II: the oxidative sequence, which is the first ATP-generating event Phase III: the second ATP-generating event

© 2012 Pearson Education, Inc. Phase I: Preparation and Cleavage The net result of the first three reactions is to convert glucose into a doubly phosphorylated molecule (fructose-1,6-bisphosphate) The phosphates are transferred to glucose from ATP ATP hydrolysis is also the driving force that makes the phosphorylation exergonic and thus irreversible

© 2012 Pearson Education, Inc. The first reaction adds a phosphate to the sixth carbon atom The bond formed is a phosphodiester bond, a lower-energy bond than the phosphoanhydride bonds in ATP The enzyme that catalyzes the reaction is hexokinase, and is specific for phosphorylation of other six-carbon sugars as well Liver cells also have glucokinase, which is specific for just glucose

© 2012 Pearson Education, Inc. The second phosphate is added to carbon one The first carbon of glucose is not as easily phosphorylated as the sixth The Gly-2 reaction first converts glucose-6- phosphate to fructose-6-phosphate (isomerase), allowing the Gly-3 reaction to add a phosphate to carbon one This reaction is catalyzed by the enzyme phosphofructokinase-1 (PFK-1)

© 2012 Pearson Education, Inc. Summary of Gly-1 to Gly-5 Glucose is split into two 3-C compounds by aldolase The first phase of the glycolytic pathway can be summarized as

© 2012 Pearson Education, Inc. Phase 2: Oxidation and ATP Generation The net energy yield of phase one is negative Two molecules of ATP have been consumed per molecule of glucose In phase 2, ATP production is linked to an oxidative event, followed by the generation of ATP in phase 3

© 2012 Pearson Education, Inc. Gly-6 and Gly-7 The oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate is highly exergonic, and drives – the reduction of NAD + to NADH (Gly-6) –the phosphorylation of ADP with inorganic phosphate, P i (Gly-7)

© 2012 Pearson Education, Inc. Important features of Gly-6 and Gly-7 NAD + is an electron acceptor The oxidation is coupled to the formation of a high-energy, doubly phosphorylated intermediate, 1,3-bisphosphoglycerate ATP generation by transferring a phosphate group to ADP from a phosphorylated substrate such as 1,3-bisphosphoglycerate is called substrate-level phosphorylation

© 2012 Pearson Education, Inc. Summary of Gly-6 and Gly-7 Gly-6 and Gly-7 can be summarized as Each reaction involving glyceraldehyde-3- phosphate occurs twice per starting molecule of glucose The two ATPs invested in the first phase are recovered in the second phase, for no net ATP

© 2012 Pearson Education, Inc. Phase 3: Pyruvate Formation and ATP Generation The phosphoester bond of 3-phosphoglycerate is converted to a phosphoenol bond The phosphate group is moved to the adjacent carbon, forming 2-phosphoglycerate (Gly-8) Water is removed from the 2-phosphoglycerate by the enzyme enolase (Gly-9) generating the high- energy compound phosphoenolpyruvate (PEP)

© 2012 Pearson Education, Inc. Phosphoenolpyruvate Hydrolysis of the phosphoenol bond of PEP is one of the most exergonic known in biological systems PEP hydrolysis drives ATP synthesis by transferring a phosphate to ADP, catalyzed by the enzyme pyruvate kinase (Gly-10) The transfer is irreversible in the direction of pyruvate and ATP formation

© 2012 Pearson Education, Inc. Summary of Gly-8 to Gly-1 The third phase of glycolysis can be summarized as

© 2012 Pearson Education, Inc. Summary of Glycolysis The two molecules of ATP formed in the second phosphorylation event (Gly-10) represent the net yield of ATP for the glycolytic pathway. The pathway is highly exergonic in the direction of pyruvate formation;  G in a cell is typically –20 kcal/mol

© 2012 Pearson Education, Inc. Conservation of Glycolysis The glycolytic pathway is one of the most common and highly conserved metabolic pathways known Virtually all cells have the ability to convert glucose to pyruvate, extracting energy in the process The next steps depend on the availability of oxygen

© 2012 Pearson Education, Inc. Alternative Substrates for Glycolysis Glucose is a major substrate for both fermentations and respiration in a variety of organisms and some tissues But for some organisms and tissues, glucose is not significant at all There are a variety of alternatives to glucose, which are often converted into an intermediate in the glucose catabolism pathway

© 2012 Pearson Education, Inc. Other Sugars and Glycerol Are Also Catabolized by the Glycolytic Pathway Many sugars are available to cells, either monosaccharides or disaccharides that can be readily hydrolyzed into monosaccharides The monosaccharides are then converted into a glycolytic intermediate Glucose and fructose enter most directly after phosphorylation on carbon atom 6; mannose and fructose require more steps

© 2012 Pearson Education, Inc. Pentoses and glycerol can be channeled into the glycolytic pathway too Phosphorylated pentoses can enter the glycolytic pathway but must first be converted to hexose phosphates Glycerol, a three-carbon molecule resulting from lipid breakdown, can enter the cycle

© 2012 Pearson Education, Inc. Figure 9-9

© 2012 Pearson Education, Inc. Polysaccharides Are Cleaved to Form Sugar Phosphates That Also Enter the Glycolytic Pathway Glucose occurs primarily in the form of storage polysaccharides, most often starch in plants and glycogen in animals

© 2012 Pearson Education, Inc. Gluconeogenesis The process of glucose synthesis is called gluconeogenesis Glucose is synthesized from three- and four- carbon precursors (noncarbohydrate) Pyruvate and lactate are the most common starting materials Occurs in all organisms and in animals mainly in liver and kidneys

© 2012 Pearson Education, Inc. Gluconeogenesis and glycolysis Gluconeogenesis occurs by simple reversal of glycolysis using the same enzyme in both directions But not all the steps are simple reversals of glycolysis: Gly-1, Gly-3, and Gly-10 are accomplished by other means These are the most exergonic reactions of glycolysis Thermodynamically difficult to reverse

© 2012 Pearson Education, Inc.

Gluconeogenesis Biosynthetic anabolic pathways are seldom just a reversal of the corresponding catabolic pathways Glucose to pyruvate has G about -20kcal/mole Reverse process would be about +20kcal/mole and highly endergonic and thermodynamically impossible

© 2012 Pearson Education, Inc. Gluconeogenesis Gly1, Gly3 and Gly10 do not simply run in reverse –Gluconeogenic pathway has bypass reactions –Gly1 and Gly3 you have hydrolytic cleaving that liberated inorganic phosphate instead of trying to synthesize ATP. Exergonic reaction with G= -3.3 kcal/mole

© 2012 Pearson Education, Inc. Gluconeogenesis Gly 10 is bypassed by a two-reaction sequence –Both reactions are driven by hydrolysis of a phosphoanydride bond –One from ATP and one from GTP –Carbon dioxide is added to pyruvate in a carboxylation reaction –Next, carboxyl group is removed by decarboxylation to form PEP

© 2012 Pearson Education, Inc. Glycolytic vs Gluconeogenesis Glycolysis is catabolic producing net two ATPs per glucose Gluconeogenesis in anabolic requiring the equivalent of six ATPs per molecule of glucose synthesized Gluconeogenesis proceed exergonically in the direction of glucose synthesis

© 2012 Pearson Education, Inc. What is happening right now to the sugars you consume? Enzymes begin to break down carbohydrates in the mouth (sucrase, maltase, lactase, etc) Glucose, galactose, and fructose are absorbed by intestinal epithelial cells.- enter bloodstream –Galactosemia Main sugar in blood is glucose. Regulated

© 2012 Pearson Education, Inc. Cori Cycle Skeletal muscle is main source of blood lactate It can function in either the presence or the absence of oxygen Liver is primary site of gluconeogenesis Lactate is converted to glucose and released into the blood-Cori cycle

© 2012 Pearson Education, Inc. The Regulation of Glycolysis and Gluconeogenesis Cells have enzymes for both glycolysis and gluconeogenesis, so the processes must be regulated Spatial regulation keeps the two processes confined to separate places in the body –Muscle cells – glycolysis Liver- gluconeogenesis There is also temporal regulation in which the two processes take place at different times in one cell

© 2012 Pearson Education, Inc. Regulation of Glycolysis and Gluconeogenesis Both are regulated to function at rates that are responsive to cellular and organismal needs for their products. ATP or glucose Regulated in reverse manner Intracellular conditions know to stimulate one pathway usually have an inhibitory effect on the other.

© 2012 Pearson Education, Inc. Key Enzymes in the Glycolytic and Gluconeogenic Pathways Are Subject to Alllosteric Regulation Allosteric regulation involves the interconversion of an enzyme between two forms, one catalytically active and the other inactive The enzyme will be active or not depending on whether an allosteric effector is bound to the allosteric site The effector might be an activator or inhibitor

© 2012 Pearson Education, Inc. Figure 9-12

© 2012 Pearson Education, Inc. Key regulatory enzymes of glycolysis and gluconeogenesis For glycolysis the enzymes are hexokinase, phosphofructokinase-1 (PFK-1), and pyruvate kinase For gluconeogenesis they are fructose-1,6- bisphosphatase, and pyruvate carboxylase Each of the enzymes is unique to its pathway so the pathways can be regulated independently

© 2012 Pearson Education, Inc. Glycolysis and gluconeogenesis are reciprocally regulated AMP and acetyl CoA, the two effectors to which both pathways are sensitive, have opposite effects AMP activates glycolysis and inhibits gluconeogenesis –Low ATP and high AMP cell is low on energy. Activate glycolysis. High ATP inhibit glycolysis.

© 2012 Pearson Education, Inc. Acetyl CoA activate gluconeogenesis but inhibits glycolysis High Acetyl CoA has enough energy inhibit glycolysis

© 2012 Pearson Education, Inc. Fructose-2,6-Bisphosphate Is an Important Regulator of Glycolysis and Gluconeogenesis Fructose-2,6-Bisphosphate (F2,6BP) is the most important regulator of both glycolysis and gluconeogenesis- ATP phosphorylation of C-2 Synthesis of F2,6BP is catalyzed by phosphofructokinase-2 (PFK-2), not PFK-1 F2,6BP activates the glycolytic enzyme (PFK-1) that phosphorylates fructose-6-phosphate and it inhibits FBPase that catalyzes the reverse reaction

© 2012 Pearson Education, Inc. Figure 9-13

© 2012 Pearson Education, Inc. Effect of cAMP on F2,6BP cAMP affects the F2,6BP concentration in two ways 1. It inactivates the PFK-2 kinase activity 2. It stimulates the F2,6BP phosphatase activity These two effects tend to decrease the concentration of F2,6BP in the cell

© 2012 Pearson Education, Inc. Effect of cAMP on hormone regulation cAMP level in cells is controlled primarily by the hormones glucagon and epinephrine (adrenaline) These cause an increase in cAMP concentration, stimulating gluconeogenesis when more glucose is needed

© 2012 Pearson Education, Inc. Novel Roles for Glycolytic Enzymes Glycolysis is connected to other cell processes Hexokinase (Gly-1) is a transcriptional repressor in yeast cells under high glucose levels Mammals have four isoforms of hexokinase -One is expressed in highly catabolically active tumor cells -Another binds to mitochondria and helps coordinate glycolysis with mitochondrial functions Many other examples exist

© 2012 Pearson Education, Inc. Additional functions of other glycolytic enzymes Glyceraldehyde-3-phosphate dehydrogenase (Gly-6) and enolase (Gly-9) have DNA-binding abilities They can act as transcriptional regulators They connect the glycolytic pathway with processes such as cell division and programmed cell death

© 2012 Pearson Education, Inc. Cancer connections Phosphoglucoisomerase (PGI; Gly-2) is involved in cell motility and migration during cancer cell metastasis Metastasis: the release of cells from malignant tumors into the bloodstream; these can form secondary tumors throughout the body PGI stimulates cell proliferation and migration