Biochemistry Lecture 11

Gluconeogenesis FIGURE 14-15 Carbohydrate synthesis from simple precursors. The pathway from phosphoenolpyruvate to glucose 6-phosphate is common to the biosynthetic conversion of many different precursors of carbohydrates in animals and plants. The path from pyruvate to phosphoenolpyruvate leads through oxaloacetate, an intermediate of the citric acid cycle, which we discuss in Chapter 16. Any compound that can be converted to either pyruvate or oxaloacetate can therefore serve as starting material for gluconeogenesis. This includes alanine and aspartate, which are convertible to pyruvate and oxaloacetate, respectively, and other amino acids that can also yield three- or four-carbon fragments, the so-called glucogenic amino acids (Table 14-4; see also Figure 18-15). Plants and photosynthetic bacteria are uniquely able to convert CO2 to carbohydrates, using the glyoxylate cycle (p. 639). 2

FIGURE 14-16 Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14-19 illustrates an alternative route for oxaloacetate produced in mitochondria. 3

Gluconeogenesis -Metabolic Pathways are Irreversible ∆G between the 1st & last metabolite is large & neg. - If 2 metabolites are interconvertible (metab 1 metab 2), the path from Metab 1  Metab 2 must be different from that of Metab 2  Metab 1 A B Metab1 Metab2 Y X

FIGURE 14-16 (part 2) Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14-19 illustrates an alternative route for oxaloacetate produced in mitochondria. 6

Carboxylation of pyruvate to oxaloacetate √ A. Circumventing Pyruvate Kinase - Conversion of Pyruvate to Phosphoenolpyruvate Carboxylation of pyruvate to oxaloacetate √ Transport of oxaloacetate out of mitochondria Oxaloacetate cyto mito Oxaloacetate NADH + H+ NADH + H+ NAD+ NAD+ Malate Malate Inner mito. Memb. 3. PEP Carboxykinase: decarboxylates and adds phosphate

FIGURE 14-17a Synthesis of phosphoenolpyruvate from pyruvate FIGURE 14-17a Synthesis of phosphoenolpyruvate from pyruvate. (a) In mitochondria, pyruvate is converted to oxaloacetate in a biotin-requiring reaction catalyzed by pyruvate carboxylase. 8

FIGURE 14-17b Synthesis of phosphoenolpyruvate from pyruvate FIGURE 14-17b Synthesis of phosphoenolpyruvate from pyruvate. (b) In the cytosol, oxaloacetate is converted to phosphoenolpyruvate by PEP carboxykinase. The CO2 incorporated in the pyruvate carboxylase reaction is lost here as CO2. The decarboxylation leads to a rearrangement of electrons that facilitates attack of the carbonyl oxygen of the pyruvate moiety on the γ phosphate of GTP. 9

FIGURE 14-16 (part 1) Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14-19 illustrates an alternative route for oxaloacetate produced in mitochondria. 10

B. Circumventing PFK – dephosphorylation of F1,6BP Dephosphorylation is not phosphorylation in reverse! Reverse Phosphorylation of ADP by F1,6 BP to generate F6P (and ATP) would be steeply uphill: F1,6 BP + ADP F6P + ATP ∆G° = +3.4 kcal/mol Instead, dephosphorylation is carried out: F1,6 BP + H2O F6P + PO4 ∆G° = -3.9 kcal/mol Reverse Phosphorylation would be mediated by PFK Dephosphorylation is mediated by F1,6BPase

C. Circumventing Hexokinase – dephosphorylation of G6P Mediated by G6Pase G6Pase is present only in liver and kidney Hence, these are the only tissues that can synthesize and secrete glucose into the blood

The Gluconeogenic Response is Activated Largely by the State of Feeding/Fasting Glycogen Blood Glucose Blood Glucose Glucose Blood Glucose Pyruvate Alanine Acetyl CoA FA’s

FIGURE 14-19 Alternative paths from pyruvate to phosphoenolpyruvate FIGURE 14-19 Alternative paths from pyruvate to phosphoenolpyruvate. The relative importance of the two pathways depends on the availability of lactate or pyruvate and the cytosolic requirements for NADH for gluconeogenesis. The path on the right predominates when lactate is the precursor, because cytosolic NADH is generated in the lactate dehydrogenase reaction and does not have to be shuttled out of the mitochondrion (see text). 14

The Cory Cycle FIGURE 23-20 Metabolic cooperation between skeletal muscle and the liver: the Cori cycle. Extremely active muscles use glycogen as energy source, generating lactate via glycolysis. During recovery, some of this lactate is transported to the liver and converted to glucose via gluconeogenesis. This glucose is released to the blood and returned to the muscles to replenish their glycogen stores. The overall pathway (glucose → lactate → glucose) constitutes the Cori cycle. 15

Gluconeogenesis & Glycolysis can Occur at the Same Time in Different Organs IN MUSCLE IN LIVER Glucose Glucose B L OO D Gluconeogen. Glycolysis Pyruvate Pyruvate Alanine Alanine Lactate Lactate RED BLOOD CELLS Glucose *** The main substrate here is LACTATE*** Pyruvate Lactate

Regulation of Metabolism

Overview of Energy Metabolism FATS POLYSACCHARIDES PROTEINS Stage I Digestion Fatty Acids, Glucose and Amino Acids Glycerol other sugars Stage II Anaerobic Acetyl CoA ATP ADP CoA Stage III Aerobic TCA cycle O2 e- CO2 Oxidative Phosphorylation

FIGURE 15-1 Metabolism as a three-dimensional meshwork FIGURE 15-1 Metabolism as a three-dimensional meshwork. A typical eukaryotic cell has the capacity to make about 30,000 different proteins, which catalyze thousands of different reactions involving many hundreds of metabolites, most shared by more than one "pathway." This overview image of metabolic pathways is from the online KEGG (Kyoto Encyclopedia of Genes and Genomes) PATHWAY database (www.genome.ad.jp/kegg/pathway/map/map01100.html). Each area can be further expanded for increasingly detailed information, to the level of specific enzymes and intermediates. 19

Principles of Regulation The flow of metabolites through the pathways is regulated to maintain homeostasis Sometimes, the levels of required metabolites must be altered very rapidly Need to increase the capacity of glycolysis during the action Need to reduce the capacity of glycolysis after the action Need to increases the capacity of gluconeogenesis after successful action

Rates of a Biochemical Reaction Rates of a biochemical reaction depend on many factors Concentration of reactants Activity of the catalyst Concentration of the enzyme Intrinsic activity of the enzyme Concentrations of effectors Allosteric regulators Competing substrates pH, ionic environment Temperature

FIGURE 15-2 Factors affecting the activity of enzymes FIGURE 15-2 Factors affecting the activity of enzymes. The total activity of an enzyme can be changed by altering the number of its molecules in the cell, or its effective activity in a subcellular compartment (1 through 6), or by modulating the activity of existing molecules (7 through 10), as detailed in the text. An enzyme may be influenced by a combination of such factors. 22

Reactions Far From Equilibrium are Common Points of Regulation

FIGURE 14-16 Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14-19 illustrates an alternative route for oxaloacetate produced in mitochondria. 24

Hexokinase Isozymes are different enzymes that catalyze the same reaction They typically share similar sequences Their regulation is often different

eg. G6P is structurally similar to glucose, and competes with glucose for active site of hexokinase

FIGURE 14-16 Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14-19 illustrates an alternative route for oxaloacetate produced in mitochondria. 27

FIGURE 15-15 Regulation of fructose 1,6-bisphosphatase (FBPase-1) and phosphofructokinase-1 (PFK-1). The important role of fructose 2,6-bisphosphate in the regulation of this substrate cycle is detailed in subsequent figures.

PFK Allosteric site AMP F6P ATP Active site

Fructose-2,6-bisphosphate

FIGURE 15-16c Role of fructose 2,6-bisphosphate in regulation of glycolysis and gluconeogenesis. Fructose 2,6-bisphosphate (F26BP) has opposite effects on the enzymatic activities of phosphofructokinase-1 (PFK-1, a glycolytic enzyme) and fructose 1,6-bisphosphatase (FBPase-1, a gluconeogenic enzyme). (c) Summary of regulation by F26BP.

FIGURE 15-17b Regulation of fructose 2,6-bisphosphate level FIGURE 15-17b Regulation of fructose 2,6-bisphosphate level. (b) Both enzyme activities are part of the same polypeptide chain, and they are reciprocally regulated by insulin and glucagon.

FIGURE 14-16 Opposing pathways of glycolysis and gluconeogenesis in rat liver. The reactions of glycolysis are on the left side, in red; the opposing pathway of gluconeogenesis is on the right, in blue. The major sites of regulation of gluconeogenesis shown here are discussed later in this chapter, and in detail in Chapter 15. Figure 14-19 illustrates an alternative route for oxaloacetate produced in mitochondria. 33

FIGURE 15-19 Regulation of pyruvate kinase FIGURE 15-19 Regulation of pyruvate kinase. The enzyme is allosterically inhibited by ATP, acetyl-CoA, and long-chain fatty acids (all signs of an abundant energy supply), and the accumulation of fructose 1,6-bisphosphate triggers its activation. Accumulation of alanine, which can be synthesized from pyruvate in one step, allosterically inhibits pyruvate kinase, slowing the production of pyruvate by glycolysis. The liver isozyme (L form) is also regulated hormonally. Glucagon activates cAMP-dependent protein kinase (PKA; see Figure 15-35), which phosphorylates the pyruvate kinase L isozyme, inactivating it. When the glucagon level drops, a protein phosphatase (PP) dephosphorylates pyruvate kinase, activating it. This mechanism prevents the liver from consuming glucose by glycolysis when blood glucose is low; instead, the liver exports glucose. The muscle isozyme (M form) is not affected by this phosphorylation mechanism.

Two Alternative Fates for Pyruvate Pyruvate can be a source of new glucose Store energy as glycogen Generate NADPH via pentose phosphate pathway Pyruvate can be a source of acetyl-CoA Store energy as body fat Make ATP via citric acid cycle Acetyl-CoA stimulates glucose synthesis by activating pyruvate carboxylase

Pancreas Adrenal Medulla Glucagon Epinephrine Muscle Liver Brain + Glucagon Epinephrine Muscle Liver Brain Glycogen Glycogen + + + + Glucose (Blood) Glucose Glucose + F6P F2,6BP F6P F2,6BP + + PFK PFK F1,6BP F1,6BP PK PK Pyruvate Pyruvate