Chapter 18 Glycolysis: Biochemistry PDF

Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett Chapter 18 Glycolysis © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed with a certain product or service or other...

Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett Chapter 18 Glycolysis © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed with a certain product or service or otherwise on a password-protected website for classroom use. Chapter 18 “Living organisms, like machines, conform to the law of conservation of energy, and must pay for all their activities in the currency of catabolism.” Ernest Baldwin Dynamic Aspects of Biochemistry Louie Pasteur’s scientific investigations into fermentation of grape sugar were pioneering studies of glycolysis. Essential Question What is the chemical basis and logic for glycolysis, the central pathway of metabolism; that is, how does glycolysis work? A Metabolic Map Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? Glycolysis Nearly every living cell carries out a catabolic process known as glycolysis - the stepwise degradation of glucose (and other simple sugars). Glycolysis is a paradigm of metabolic pathways. Carried out in the cytosol of cells, it is basically an anaerobic process; its principal steps occur with no requirement for oxygen. 18.1 What Are the Essential Features of Glycolysis? Glycolysis is also called the Embden-Meyerhof (or Warburg) Pathway Essentially all cells carry out glycolysis Ten reactions – essentially the same in all cells but with different rates 18.1 What Are the Essential Features of Glycolysis? Two phases: o First phase converts glucose to two molecules of glyceraldehyde-3-P o Second phase produces two pyruvates Products are pyruvate, ATP, and NADH There are three possible fates for pyruvate Figure 18.1 The Glycolysis Pathway This is the figure I will use for the quiz Mindtap The Fates of Pyruvate From Glycolysis Figure 18.2 Pyruvate produced in glycolysis can be used by cells in several ways. In animals, pyruvate is normally converted to acetyl-coenzyme A, which is then oxidized in the TCA cycle to produce CO2. When oxygen is limited, pyruvate can be converted to lactate. Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2. Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? 18.2 – Why Are Coupled Reactions Important in Glycolysis? Conversion of glucose to two lactates: energy producing (-183.6 kJ/mol) Conversion of two ADPs to two ATPs: energy requiring (61 kJ/mol) These two reactions are coupled so that the energy released from the conversion of glucose to lactate can be used to drive the synthesis of ATP from ADP. Conversion of one molecule of glucose to pyruvate in glycolysis drives the production of two molecules of ATP. 18.2 Why Are Coupled Reactions Important in Glycolysis? Coupled reactions convert some, but not all, of the metabolic energy of glucose into ATP Under cellular conditions, approximately 50% of the available energy in glucose is converted into ATP. Coupled reactions involving ATP hydrolysis are also used to drive the glycolytic pathway Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? Chemical Principles and Features of the First Phase of Glycolysis In the first phase of glycolysis, glucose is converted into two molecules of glyceraldehyde-3-phosphate. o Glucose is phosphorylated to glucose-6-P, which is isomerized to fructose-6-P. o Another phosphorylation and then cleavage yields two three-carbon intermediates. o One of these is glyceraldehyde-3-P, and the other, dihydroxyacetone-P, is converted to glyceraldehyde3-P. Energy released from this high-energy molecule in the second phase of glycolysis is then used to synthesize ATP. Reactions and Thermodynamics of Glycolysis Compare to Reaction 1 in Fig. 18.1! Figure 18.1 The Glycolysis Pathway Mechanism Handout: Hexokinase/Glucokinase What Are the Chemical Principles & Features of the 1st Phase of Glycolysis? The first reaction - phosphorylation of glucose Hexokinase or glucokinase This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous Be sure you can interconvert Keq and standard-state free energy change Be sure you can use Eq. 3.8 to generate the values shown in the far-right column of Table 18.1. Hexokinase Primes the Pump for Glycolysis Figure 18.3 Just as a water pump must be “primed” with water to get more water out, the glycolytic pathway is primed with ATP in steps 1 and 3 in order to achieve net production of ATP in the second phase of the pathway. Rxn 1: Hexokinase 1st step in glycolysis; ∆G large, negative Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell Glucose Is Kept in the Cell by Phosphorylation to Glucose-6-Phosphate Figure 18.4 Glucose-6-P cannot cross the plasma membrane. Glucose-6-P Is Common to Several Metabolic Pathways Figure 18.5 Glucose-6-phosphate is the branch point for several metabolic pathways. Figure 18.1 The Glycolysis Pathway Mechanism Handout: Phosphoglucoisomerase Reaction 2: Phosphoglucoisomerase Glucose-6-P to fructose-6-P Why does this reaction occur? o Next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH o Isomerization activates C-3 for cleavage in aldolase reaction Ene-diol intermediate in this reaction Be able to write a mechanism for this reaction (see Figure 18.8) Figure 18.1 The Glycolysis Pathway Mechanism Handout: Phosphofructokinase Reaction 3: Phosphofructokinase PFK is the committed step in glycolysis! The second priming reaction of glycolysis Committed step and large, negative ΔG – means PFK is highly regulated ATP is both a substrate and an allosteric inhibitor; AMP activates Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator Reaction 3: Phosphofructokinase PFK increases activity when energy status is low PFK decreases activity when energy status is high Phosphofructokinase Behaves Cooperatively at High [ATP] Figure 18.9 At high ATP, phosphofructokinase (PFK) behaves cooperatively, and the activity plot is sigmoid. Phosphofructokinase – A Second Phosphorylation Driven by ATP Phosphofructokinase is the second “priming” reaction of glycolysis. ATP is consumed in this priming reaction, so that more ATP can be produced further along the pathway. F-2,6-BP Regulates Phosphofructokinase Phosphofructokinase is regulated by fructose-2,6bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-P. Phosphofructokinase Is Activated by F2,6-BP Figure 18.10 Fructose-2,6bisphosphate activates phosphofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate concentration. Figure 18.1 The Glycolysis Pathway Mechanism Handout: Fructose Bisphosphate Aldolase Reaction 4: Aldolase A six-carbon intermediate is cleaved to two threecarbon intermediates (DHAP and G-3-P) Animal aldolases are Class I aldolases In Class I aldolases, a covalent Schiff base intermediate is formed between the substrate and an active-site lysine The evidence for a Schiff base intermediate for Class I aldolases is described in Problem 18 on page 641 The Fructose Bisphosphate Aldolase Reaction ΔG = -0.23 kJ/mol The aldolase reaction is unfavorable as written at standard state. The cellular ΔG, however, is close to zero. Figure 18.1 The Glycolysis Pathway Mechanism Handout: Triose Phosphate Isomerase Reaction 5: Triose Phosphate Isomerase DHAP is converted to G-3-P This reaction makes it possible for both products of the aldolase reaction to continue in glycolysis The reaction involves an ene-diol mechanism Glu165 in the active site acts as a general base Triose phosphate isomerase is a near-perfect enzyme - see Table 13.5 on page 450 Triose Phosphate Isomerase Mechanism Figure 18.13 A reaction mechanism for triose phosphate isomerase. In the yeast enzyme, the catalytic residue is Glu165. Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? Figure 18.1 The Glycolysis Pathway What Are the Chemical Principles & Features of the 2nd Phase of Glycolysis? Metabolic energy of glucose produces four ATP Net ATP yield for glycolysis is two ATP The second phase of glycolysis involves two very high-energy phosphate intermediates o 1,3-bisphosphoglycerate (1,3-BPG) o Phosphoenolpyruvate (PEP) Figure 18.1 The Glycolysis Pathway Mechanism Handout: Glyceraldehyde-3Phosphate Dehydrogenase Reaction 6: Glyceraldehyde-3-P Dehydrogenase G-3-P is oxidized to 1,3-BPG Energy yield from converting an aldehyde to a carboxylic acid is used to make 1,3-BPG and NADH The mechanism involves covalent catalysis and a nicotinamide coenzyme, and it is good example of nicotinamide chemistry This enzyme reaction is the site of action of arsenate – an anion analogous to phosphate G3P-DH Is the Site of Action of Arsenate Arsenate is a substrate for the G3P-DH reaction, forming 1-arseno-3-phosphoglycerate. This product breaks down to 3-phosphoglycerate, essentially bypassing the phosphoglycerate kinase reaction. The result is that glycolysis in the presence of arsenate produces no net ATP. Figure 18.1 The Glycolysis Pathway Mechanism Handout: Phosphoglycerate Kinase Reaction 7: Phosphoglycerate Kinase ATP synthesis from a high-energy phosphate Phosphoglycerate kinase transfers a phosphoryl group from 1,3-bisphosphoglycerate to ADP to form an ATP. This is referred to as “substrate-level phosphorylation” This reaction “pays off” the ATP debt created by the priming reactions in the first phase 2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction (Figure 18.15) Figure 18.1 The Glycolysis Pathway Mechanism Handout: Phosphoglycerate Mutase Reaction 8: Phosphoglycerate Mutase Phosphoglycerate mutase catalyzes a phosphoryl group transfer from C-3 to C-2 Rationale for this reaction in glycolysis: It repositions the phosphate to make PEP in the following reaction (enolase) Note the phospho-histidine intermediate Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a bit of 2,3-BPG is required to phosphorylate the active-site histidine Nomenclature note: a “mutase” catalyzes migration of a functional group within a substrate Example Problem Describe the reaction and mechanism catalyzed by phosphoglycerate mutase. What other molecules are required for activity? Figure 18.1 The Glycolysis Pathway Mechanism Handout: Enolase Reaction 9: Enolase Conversion of 2-phosphoglycerate to PEP Enolase makes a high-energy phosphate in preparation for ATP synthesis in step 10 The overall ΔG for this reaction is 1.8 kJ/mol How can such a reaction create a PEP? "Energy content" of 2-PG and PEP are similar Enolase just rearranges 2-PG to a form that releases more energy upon hydrolysis Figure 18.1 The Glycolysis Pathway Mechanism Handout: Pyruvate Kinase Reaction 10: Pyruvate Kinase Conversion of PEP to pyruvate makes ATP These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis Large, negative ΔG – indicating that this reaction is subject to regulation PK is allosterically activated by AMP and F-1,6bisP PK is allosterically inhibited by ATP and acetylCoA Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? Metabolic Fates of NADH and Pyruvate Produced in Glycolysis In addition to ATP, the products of glycolysis are NADH and pyruvate. Their processing depends upon other cellular pathways. NADH can be recycled by both aerobic and anaerobic paths, either of which results in further metabolism of pyruvate. What a given cell does with the pyruvate produced in glycolysis depends in part on the availability of oxygen. What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? NADH can be recycled via aerobic or anaerobic pathways NADH represents energy - two possible fates: o If O2 is available (aerobic conditions), NADH is oxidized in the electron transport pathway, making ATP in oxidative phosphorylation o In anaerobic conditions, NADH is oxidized by lactate dehydrogenase (LDH) or alcohol dehydrogenase (ADH), providing additional NAD+ for more glycolysis Anaerobic Pyruvate Reduction to Ethanol in Yeast Regenerates NAD+ Figure 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (Right) Fermentation at a bourbon distillery. A “mash” of corn and other grains is fermented by yeast, producing ethanol and CO2, which can be seen bubbling to the surface. Anaerobic Pyruvate Reduction to Lactate in Animals Regenerates NAD+ Figure 18.21 (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction. (Right) Strenuous exercise. Also, hibernating turtles become “anoxic” and convert glucose mainly to lactate. Their shells release minerals to buffer the lactate throughout the period of hibernation. Mindtap What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? Pyruvate also represents energy - two possible fates: o Aerobic or anaerobic paths In aerobic conditions, pyruvate proceeds through the tricarboxylic acid (TCA) cycle (see Chapter 19) Anaerobic metabolism of pyruvate leads to lactate (in microorganisms and animals) or ethanol (in yeast) These are examples of fermentation – the production of ATP energy by reaction pathways in which organic molecules function as donors and acceptors of electrons Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? 18.6 How Do Cells Regulate Glycolysis? The elegant evidence of regulation See Figure 18.22 Standard state ΔGo´ values provide little insight into the actual free energy changes that occur in glycolysis The plot of ΔG values in cells is revealing: o Most values are near zero o Three of ten reactions have large, negative values 18.6 How Do Cells Regulate Glycolysis? These three reactions with large negative ΔG are sites of regulation (hexokinase, phosphofructokinase, and pyruvate kinase) Regulation of these three reactions can turn glycolysis off and on 18.6 How Do Cells Regulate Glycolysis, Con’t? The plot of ∆G values in cells is revealing, con’t: o Three of ten reactions have large, negative ∆G (hexokinase, phosphofructokinase, and pyruvate kinase).  When these three enzymes are active, glycolysis proceeds, and glucose is readily metabolized to pyruvate.  Inhibition of these three enzymes brings glycolysis to a halt.  Large negative ∆G reactions are sites of regulation. Cellular Free Energies of the Reactions of Glycolysis 1=hexokinase Figure 18.22 The free energies of the reactions of glycolysis under actual intracellular concentrations of metabolites in erythrocytes. 3=phosphofructokinase 10=pyruvate kinase 11=lactate dehydrogenase Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? 18.7 Are Substrates Other Than Glucose Used in Glycolysis? Sugars other than glucose can be glycolytic substrates Dietary fructose is degraded almost exclusively in the liver in an unregulated process that circumvents the PFK reaction (Figure 18.23a) Mannose is routed into glycolysis by conventional means (Figure 18.23b) Galactose is more interesting - the Leloir pathway "converts" galactose to glucose - sort of.... See Figure 18.23 Fructose Metabolism Figure 18.23a Fructose metabolism in the liver is unregulated. Aldolase B, a class I aldolase unique to the liver, converts fructose-1-P to glyceraldehyde, which must be phosphorylated by triose kinase to continue through the glycolytic pathway. Fructose that reaches kidneys or muscle: Hexokinase converts fructose to F-6-P. Fatty liver disease and cirrhosis Mannose and Galactose Can Enter Glycolysis Figure 18.23b Mannose (and dietary fructose that reaches the kidneys and muscle) and galactose can enter the glycolytic pathway via hexokinase ahead of the phosphofructokinase reaction. Thus, their catabolism is subject to the same regulation as glucose. The Leloir Pathway Figure 18.24 Galactose metabolism via the Leloir pathway. Glycerol Can Also Enter Glycolysis Glycerol is produced in the decomposition of triacylglycerols. It can be converted to glycerol-3-P by glycerol kinase. Glycerol-3-P is then oxidized to dihydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase (next slide). Glycerol Can Also Enter Glycolysis Glycerol is produced in the decomposition of triacylglycerols. It can be converted to glycerol-3-P by glycerol kinase (previous slide). Glycerol-3-P is then oxidized to dihydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase. Outline 18.1 What are the essential features of glycolysis? 18.2 Why are coupled reactions important in glycolysis? 18.3 What are the chemical principles and features of the first phase of glycolysis? 18.4 What are the chemical principles and features of the second phase of glycolysis? 18.5 What are the metabolic fates of NADH and pyruvate produced in glycolysis? 18.6 How do cells regulate glycolysis? 18.7 Are substrates other than glucose used in glycolysis? 18.8 How do cells respond to hypoxic stress? (on your own) Do You Understand… 1. Glycolysis? 2. The fate of pyruvate?