Chapter 15 Homework Assignment

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1 Chapter 15 Homework Assignment The following problems will be due once we finish the chapter: 3, 5, 6, 8, 9 Chapter 15 1 Regulation of Metabolic Pathways Dynamic Steady State Fuels, such as glucose, enter a cell and waste products, such as CO 2, leave the cell. However, the mass and gross components of the cell do not change appreciatively The cell maintains a dynamic steady state For metabolic pathways, this means that as a product is produced by one reaction, it is used a the substrate of the next reaction at the same rate. ν A 1 ν S 2 P When ν1 = ν 2 [S] is constant Therefore, the concentration of the substrate remains constant. When this steady state is disturbed by some change in external circumstances or energy supply, the altered movement of compounds through the pathway (aka. the flux) triggers regulatory mechanisms intrinsic to each pathway. Chapter 15 3 Chapter 15 Principles of Metabolic Regulation Regulation of Metabolic Pathways Response to Metabolite Concentrations Flux through a biochemical pathway depends on the activities of the enzymes that catalyze each reaction in that pathway. For some steps, the reaction is close to equilibrium within the cell. For these reactions, small changes in the concentrations of the substrate or product can produce large changes in the net rate of the reaction, even favoring the reverse direction. These steps can be identified d by comparing the mass action ratio (Q) with the equilibrium constant (K eq ) If these two values are within a few orders of magnitude of one another, the reaction is near equilibrium If the two are not close then the reactions shifts to bring it closer to equilibrium Q > K eq System proceeds to form reactants. Q = K eq System is at equilibrium. Q < K eq System proceeds to form products Chapter

2 Regulation of Metabolic Pathways Response to Metabolite Concentrations of Glycolysis & Gluconeogenesis Free Energy, kjou ule/mole Glycolysis: Free Energies A B G G-6-P F-6-P F-1,6-bP GA-3-P G-3-P 1,3-bPG 3-PG 2-PG PEP PYR C There are three irreversible steps during Glycolysis. Each of these is an excellent point for regulation A - Hexokinase: The first step of glycolysis B Phosphofructokinase-1 (PFK-1): The committed step of glycolysis C- Pyruvate Kinase: The very last step of glycolysis Chapter 15 5 Chapter 15 7 Regulation of Metabolic Pathways Alteration of Enzyme Activity The activity of an enzyme can be modulated by changes in the number of enzyme molecules in the cell or by changes in the catalytic activity of each enzyme molecule already present. Change in the number of enzyme molecules is a relatively slow process whereas changes in the activity is a faster process. Mechanisms of flux regulation employ both reduction and enhancement of enzyme activity Regulation of activity can occur in one of four ways as shown above The upper two are used to regulate the activity of hexokinase IV (glucokinase). The lower two are combined to control pyruvate kinase, and the regulation by fructose- 2,6-bisphosphate. Isozymes (tissue-specific variants), feedback inhibition, and coordinate regulation are also employed Chapter 15 6 Isozymes An Aside Isozymes are different proteins that catalyze the same reaction These multiple forms may occur in the same species, in the same tissue, or even in the same cell The different forms generally differ in their kinetic and/or regulatory properties, in the cofactors they use or in their subcellular location Hexokinase, which catalyzes the conversion of Glucose to Glucose- 6-phosphate, is regulatory enzyme with four isozymes (Hexokinase I IV). Each of these isozymes is encoded by a different gene Hexokinases I III are found in muscle tissues (myocytes) Hexokinase IV (aka. Glucokinase) is found in liver cells (hepatocytes) The isozymes vary from one another in their K M values and their regulation Chapter

3 Hexokinase Regulation of Glycolysis Step 1 In MUSCLE Hexokinase-II K M = 0.1 mm Runs near V max unless glycolysis slows G6P will build up and thus inhibit the enzyme In LIVER Glucokinase (HK IV) K M = 10 mm Inhibition is mediated by glucokinase regulatory protein F6P stabilizes the interaction - glucose destabilizes it! Runs << V max, except after a meal rich in carbohydrate Blood glucose ~ 5 mm Chapter 15 9 PFK-1 Regulation of Glycolysis Step 3 High [ATP] signals energy needs are met; high citrate that TCA throughput is low either way, glycolysis slows Conversely, high [ADP] or [AMP] indicates more ATP is needed, and high F-2,6-BP signals high blood sugar (its levels are hormone-regulated) Chapter Glucokinase (HK IV) Regulation by Protein Association and Sequestration In the liver, glucokinase (hexokinase IV) is regulated by its high affinity for a nuclear regulator protein This complex cannot move into the cytosol where glycolysis takes place In the presence of high levels of F6P, this interaction is much tighter, keeping HK IV in the nucleus Glucose competes with F6P for binding, resulting in the release of the HK IV and its movement into the cytosol Unlike the muscle isozyme, glucokinase is not inhibited by glucose-6- phosphate Chapter Pyruvate Kinase Regulation of Glycolysis Step 3 There are at least 3 isozymes of PK found in vertebrates ATP, Acetyl CoA (first reactant of TCA) and long-chain fatty acids all signal that abundant energy is available, so that glycolysis can slow High [ATP] allosterically decreases the affinity of PK1 for PEP, raising the K M enough that the reaction is substantially slowed The liver form (L form) but not the muscle form (M form) is also subject to further regulation by phosphorylation in response to Glucagon signals Chapter

4 Gluconeogenesis Regulation The first regulatory point for gluconeogenesis is determination of the fate of pyruvate Does is move into the TCA cycle for energy production or does is get converted into glucose for export? This means regulation of two enzymes in tandem, Pyruvate dehydrogenase d (PDH) and pyruvate carboxylase (PCB) PDH is inhibited by high levels of Acetyl-CoA while PCB is positively modulated by high levels of Acetyl-CoA. Hormonal Regulation - Glucagon The liver is the overseer of blood glucose levels requiring additional regulatory mechanisms to coordinate glucose production and consumption within the hepatocytes We already know that the hormone Glucagon signals the liver to produce and release glucose into the blood. One source of glucose within the liver is within its glycogen molecules, the other source is via gluconeogenesis In addition to the enzyme cascade that leads to activation of Glycogen phosphorylase, glucagon also activates Fructose 2,6-bisphosphatase (FBPase- 2) FBPase-2 dephosphorylates fructose 2,6-bisphosphate, an allosteric effector of the enzymes PFK-1 and FBPase-1. Why should the same signal have these opposite effects? Chapter Chapter Gluconeogenesis Regulation FBPase-1 The second control point is the reaction catalyzed by FBPase-1 This reaction is inhibited by high [AMP] At the same time, high [AMP] stimulates phosphofructokinase-1 Hormonal Regulation F2,6BP Why would nature set the regulation of these reactions up in this manner? Chapter What does Glucagon signal? How does it effect the levels of F2,6BP? What do the varied levels of F2,6BP indicate? Should that favor Glycolysis or Gluconeogenesis? What hormone should have the opposite effect? Chapter

5 Hormonal Regulation F2,6BP PFK-2 and FBPase-2 are two distinct enzymatic activities of a single, bifunctional protein (one polypeptide chain, two activities!) Glucagon activates FBPase-2 activity via phosphorylation by a camp-dependent d protein kinase This activation reduces levels of the effector F2,6BP and favors gluconeogenesis Insulin has the opposite effect by stimulating the activity of a phosphoprotein phosphatase that dephosphorylates the PFK-2/FBPase-2 protein This removal activates the PFK-2 activity, producing F2,6BP, and favoring glycolysis Chapter Glycogen Metabolism Glycogen Storage In a wide range of organisms, excess glucose is stored in polymeric forms (e.g. Starch in plants and glycogen in animals) Glycogen can represent up to 10% of the weight of the liver and 1 2% of the weight of muscles. This polymeric storage keeps the concentration of glucose to ~ 0.01 µm The amount of glycogen in the muscles is there to provide a quick source of energy and can be exhausted in ~ 1 hour of vigourous exercise. In the liver, the glycogen serves as a reservoir of glucose for other tissues when dietary glucose is not available. This source can be depleted in hours Glycogen is stored in granules called β-particles which consists of up to 55K glucose residues with about 2000 reducing ends. These particles also include the enzymes needed for breakdown and synthesis as well as regulatory machinery. Chapter Alternative Uses for Glucose Glycogen Metabolism - Breakdown Breakdown Catalysis of Glycogen The glucose residues of the outer branches of glycogen enter glycolysis through the action of three enzymes: Glycogen Phosphorylase Glycogen Debranching Enzyme Phosphoglucomutase What do you think each enzyme does? Chapter Chapter

6 Glycogen Metabolism - Breakdown Glycogen Phosphorylase Glycogen phosphorylase catalyzes the phosphorolysis of the α (1 4) glycosidic bond between two glucose residues at the non-reducing end of glycogen. The bond undergoes attack by inorganic phosphate (P i ) producing Glucose-1- phophate and (Glycogen) n-1 Pyridoxal phosphate is an essential cofactor in this reaction Its phosphate acts as a general acid catalyst, promoting attack on the glycosidic bond GP acts continuously on a glycogen molecule until it reaches a point four residues away from a branch point where it stops. Further degradation can only continue after the Debranching enzyme catalyzed two successive reactions that transfer branches. Chapter Glycogen Metabolism - Breakdown Phosphoglucomutase G1P, the end product of GP, is converted to G6P by phosphoglucomutase The enzyme is initially phosphorylated at a Ser residue and donates this group to the C- 6 position of the G1P, yielding the shortlived G1,6P The enzyme then accepts a P i group from the C-1 position, regenerating the enzyme and producing G6P. In the muscle, this product goes right into glycolysis for energy production. In the liver, the purpose of this product is to move glucose into the blood when levels have decreased. Chapter Glycogen Metabolism - Breakdown Debranching Enzyme When an α (1 6) linkage is met, the phosphorylase cannot proceed A bifunctional debranching enzyme first transfers a block of 3 glucose residues to the nonreducing end of the chain by its transferase activity, Then employs its glucosidase α (1 6) activity to remove the glucose moiety at the branchpoint Glycogen phosphorylase then resumes its work on the now-linear (unbranched) polymer (until it hits the next branchpoint, if any) Glycogen Metabolism - Breakdown Glucose-6-Phosphatase Since the liver breaks down glycogen in an effort to increase blood glucose levels, the G6P must be dephosphorylated before movement into the blood. Glucose-6-phosphatase is found only in liver and kidney tissues and is responsible for this reaction The enzyme is an integral membrane protein with its active site facing the lumen of the endoplasmic reticulum (ER) Transporters move the reactants and products into and out of this area Chapter Chapter

7 Glycogen Metabolism - Synthesis Sugar Activation G1P Many of the reactions in which hexoses are transformed or polymerized involve sugar nucleotides These complexes have several properties that are useful for biosynthetic reactions The condensation reaction between a sugar phosphate (like Glu-1-P) and a nucleotide (like UTP) is irreversible ( G kj/mol due to PP i hydrolysis) The nucleotide moiety has many groups that can undergo non-covalent interactions with enzymes, adding free energy of binding to the mix The activated sugar carbon is primed for nucleophilic attack due to the presence of the excellent nucleotide leaving group The nucleotidyl tag marks such sugars for certain purposes (like glycogen synthesis), and prevents them from being used in other metabolic pathways (like glycolysis) Chapter Glycogen Metabolism - Synthesis Glycogen Synthase Making glycogen begins with G6P (from where?) being converted to G1P by phosphoglucomutase G1P is then converted in turn by UDP-glucose pyrophosphorylase into UDP-glucose UDP-Glucose is added by the enzyme glycogen synthase in α (1 4) linkage to nonreducing ends of existing glycogen as long as the α (1 4) primer has at least 8 glucose residues Chapter Glycogen Metabolism - Synthesis Initiation A 37 kd protein, glycogenin, is both the primer on which new chains are begun, and the enzyme that lengthens them Glycogenin acquires glucose by its glucosyltransferase activity from UDP-glu in a group transfer reaction; glucose becomes covalently attached to Tyr 194 Glycogen synthase then joins as a tight complex, and up to 7 more residues are added from UDPglu by glycogenin Glycogen synthase then takes over, extending the chain as it moves away from glycogenin A branching enzyme completes the glycogen particle, eventually dissociating, but glycogenin stays right there! 3eFig Chapter Glycogen Metabolism - Synthesis Glycosyl-(4 6)-transferase Problem: Glycogen synthase cannot do α (1 6) branching This is the exclusive job of glycosyl-(4 6)-transferase, which removes 6-7 residues from a strand at least 11 long These residues are transfered to the C-6 carbon of a more interior glucose of the same (or another) chain, creating a new branch! Branches make glycogen more soluble, and allow for faster removal (or addition) of glucose, since there are more ends Chapter

8 Glycogen Metabolism - Synthesis A Glycogen Particle Glycogen Metabolism - Regulation Glycogen Phosphorylase Isozymes hidden exposed About 55,000 glucose residues with a MW ~10 7 Chapter Fig The muscle and liver enzymes are isozymes: Muscle has no glucagon receptors, and the enzyme is allosterically stimulated by both AMP and calcium, present at high levels during muscle contraction Liver must mobilize glycogen breakdown for export of glucose Once blood glucose returns to normal levels, it occupies a unique site on this isozyme that both inhibits its activity and exposes Ser 14 -P to PP1 (itself activated by insulin) Chapter Glycogen Metabolism - Regulation Glycogen Phosphorylase Stimulated by glucagon or epinephrine (via a camp cascade) Phosphorylation at Ser 14 allosterically activates its ability to break down glycogen (to G1P) This is reversible by the action of phosphoprotein phosphatase (PP1) Chapter The Role of Epinephrine and Glucagon in Glucose Liberation In the liver, Glucagon is the signal that activates the cascade for the production of glucose. This peptide hormone is a signal that the body is in a starvation stage and produced in pancreatic α cells in response to low blood glucose levels. Glucagon interacts with the Glucagon serpentine receptor WHY? that is very similar to the β- Adrenergic receptor of Ephinepherine The resulting camp acts as a second messenger. Just in case you forgot! Chapter

9 Glycogen Metabolism - Regulation Glycogen Synthase Similar to GP, Glycogen Synthase can exist in either the phosphorylated or dephosphorylated form Phosphorylation of the hydroxyl side chain of several Ser residues of both subunits converts active GSa to inactive GSb This enzyme can be phosphorylated by at least 11 different protein kinases, with the most important one being Glycogen Synthase Kinase 3 (GSK3) However, the action of GSK3 is hierarchical meaning that a different kinase (Casein kinase II; CKII) must phosphorylate certain residues before GSK3 can do its job (this is called priming) Chapter Glycogen Metabolism - Regulation Effect of Insulin Are these effects logical? Chapter Glycogen Metabolism - Regulation Effect of Insulin Glycogen Metabolism - Regulation Excellent Summary Did you miss this over break? Chapter Chapter

10 Glycogen Metabolism When Things Go Bad Chapter