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Chapter 18 Glycolysis

Dr Khairul Ansari

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.

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Essential Question
What is the chemical basis and logic for glycolysis, the central pathway of metabolism; that is, how does glycolysis work? What are the essential features of glycolysis? Why are coupled reactions important in glycolysis? What are the chemical principles and features of the first phase of glycolysis? What are the chemical principles and features of the second phase of glycolysis? What are the metabolic fates of NADH and pyruvate produced in glycolysis? How do cells regulate glycolysis? Are substrates other than glucose used in glycolysis? How do cells respond to hypoxic stress?

Gycolysis
Living organism appears in O2 free environment Gycolysis does not need O2 Gycolysis plays significant roles in anaerobic metabolism in the first 2 billion years of evolution Otto Warburg, Gustav Embden and Otto Fritz Meyerhof- worked out of the glycolysis pathway Gycolysis is also known as Embden-Meyerhof (or Warburg) Pathway

Brain, Kidney medulla and rapidly contracting skeleting muscle and some cells such as erythrocytes and sperm use glucose as only energy source.

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Glycolysis
Essentially all cells carry out glycolysis Ten reactions essentially the same in all cells but with different rates Two phases: First phase converts glucose to two glyceraldehyde-3-P Second phase produces two pyruvates Products are pyruvate, ATP and NADH Three possible fates for pyruvate Under aerobic conditions pyruvate can be converted to acetyl-CoA that enter TCA cycle It can also be converted into glucose via gluconeogenesis. Under anaerobic conditions, it may be converted into lactic acid. In yeast, pyruvate is converted into ethanol instead.

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 acetylcoenzyme 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.

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The Glycolysis Pathway

Phase I:- Five reaction break down glucose to two molecules of Glyceraldehyde -3-Phosphate (G-3-P) consume two ATP Phase II:- G-3-P is converted to two molecules of pyruvate-produce 4 ATP

The Glycolysis Pathway

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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 5% of the energy of glucose is released in glycolysis Coupled reactions involving ATP hydrolysis are also used to drive the glycolytic pathway Net gain of Glycolysis is 2 ATP (61 kj/mol) and 2 pyruvate Glucose 2 molecules of lactate C6H12O6 2 H3C-CHOH-COO- + 2 H+ G = -183.6 KJ/mol The excess energy is utilized in rearrangement of the molecules Glucose + 2 ADP + 2 Pi 2 lactate + 2 ATP + 2 H+ 2 H2O G = -183.6 + 61 = - 122.6 KJ/mol Under standard condition (61/183.6)x 100 = 33% energy is stored in ATP

What Are the Chemical Principles and Features of the First 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. 3.13 to generate the values shown in the far right column of Table 18.1. -D-Glucose + ATP4- -D-Glucose -6-phosphate2- +ADP3- + H+ G = -16.7 KJ/mol Phosphorylation of glucose cost 13.8 KJ/mol

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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. Advantages of Phosphorylation 1. Plasma membrane is impermeable to -D-Glucose -6-phosphate2(negatively charged) 2. Conversion of glucose to -D-Glucose -6-phosphate2- , keeps the intracellular concentration low, favoring diffusing into the cell 3. Favorable thermodynamics of the first reaction makes it an important site for regulation

Glucose is kept in the cell by phosphorylation to glucose-6-phosphate

Figure 18.4 Glucose-6-P cannot cross the plasma membrane.

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Reactions and Thermodynamics of Glycolysis

Rxn 1: Hexokinase
1st step in glycolysis; G large, negative Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell Km for glucose is 0.1 mM; cell has 4 mM glucose So hexokinase is normally active! Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P - but is not the most important site of regulation of glycolysis - Why? Hexokinase reaction is one of the three points in glycolysis pathway that are regulated Mg2+ is required (the true substrate is MgATP2-) There are two isozymes of Hexokinase (type I and type II)

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Rxn 1: Hexokinase
Km for glucose is 0.03 mM for type I hexokinase and 0.3 mM for type II hexokinase Type I is primary hexokinase in brain Type IV (glucokinase) is predominant in liver- doest not get inhibited by the product (Km is approximately 10 mM) When glucose level is high the liver hexokinase (glucokinase) phosphorylates glucose and eventually converts to glycogen Glucokinase is inducible (by insulin) and acts when level of glucose is very high. In diabetes mellitus (patient producing insufficient insulin) level of glucokinase is less.

Steady-State Concentrations of Glycolytic Intermediates

These steady-state concentrations are used to obtain the cellular values of G found in Table 18.1 and Figure 18.22.

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Glucose-6-P is common to several metabolic pathways

Figure 18.5 Glucose-6-phosphate is the branch point for several metabolic pathways.

Reaction 1: Hexokinase

Figure 18.6 The (a) open and (b) closed states of yeast hexokinase. Binding of glucose (green) induces a conformation change that closes the active site, as predicted by Daniel Koshland. The induced fit model for enzymes is discussed on page 409 of the text.

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Hexokinase is the paradigm of induced fit

Figure 18.7 (a) Mammalian hexokinase I contains an Nterminal domain (top) and a Cterminal domain (bottom) joined by a long -helix. Each of these domains is similar in sequence and structure to yeast hexokinase. (b) Human glucokinase undergoes induced fit upon binding glucose (green).

Reaction 2: Phosphoglucoisomerase
Glucose-6-P to Fructose-6-P Why does this reaction occur? next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH isomerization activates C-3 for cleavage in aldolase reaction Ene-diol intermediate in this reaction

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A mechanism for phosphoglucoisomerase

Figure 18.8 The phosphoglucoisomerase mechanism involves opening of the pyranose ring (step 1), proton abstraction leading to enediol formation (step 2), and proton addition to the double bond, followed by ring closure (step 3)

Reaction 3: Phosphofructokinase
PFK is the committed step in glycolysis! The second priming reaction of glycolysis Committed step and large, negative G - means PFK is highly regulated ATP inhibits, AMP reverses inhibition Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator PFK increases activity when energy status is low PFK decreases activity when energy status is high The reaction is endegonic by itself (G = 16.6 KJ/mol), but exergonic when coupled with ATP hydrolysis (G = -14.2 KJ/mol

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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.

Phosphofructokinase
Phosphofructokinase is the valve controlling rate of glycolysis ATP is both substrate and allosteric regulator of PFK High ATP turn off glycolysis in cytosol. ATP level does not fluctuate to greater extend in most tissue ADP + ADP ATP + AMP Small drop of ATP level in cell results a large increase in AMP AMP reverse the PFK inhibition by ATP PFK activity is high when energy status of cell is low, and PFK activity is low when energy status is high. Fructose-2,6-bisphosphate also regulatesPFK Citrate an intermediate of TCA cycle is also an allosteric inhibitor of PFK (PFK activity coupled with TCA cycle)

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Phosphofructokinase behaves cooperatively at high [ATP]

Figure 18.9 At high ATP, phosphofructokinase (PFK) behaves cooperatively and the activity plot is sigmoid.

F-2,6-BP regulates Phosphofructokinase

Phosphofructokinase is regulated by fructose-2,6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-P.

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F-2,6-BP reverses the inhibition of PFK by ATP

Figure 18.11 F-2,6-BP stimulates PFK by decreasing the inhibitory effects of ATP.

Rxn 4: Aldolase (Fructose bisphosphate aldolase

A C6 intermediate is cleaved to 2 C3s (Dihydroxyacetone phosphate and and Glyceraldehyde-3-phosphate) The reaction is enderonic (cellular G, however, is close to zero.) The reaction rate (equilibrium ) is influenced by concentration and may become exergonic Class I and II aldolases present in animal and bacteria respectively Class II aldolase contain Zn2- in the active site

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Reaction 5: Triose Phosphate Isomerase

Triose phosphate isomerase completes the first phase of glycolysis. Each glucose has been converted to two molecules of glyceraldehyde3-phosphate.

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

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What Are the Chemical Principles and Features of the Second Phase of Glycolysis?
Metabolic energy of glucose produces 4 ATP Net ATP yield for glycolysis is two ATP The second phase of glycolysis involves two very high-energy phosphate intermediates 1,3-bisphosphoglycerate (1,3-BPG) Phosphoenolpyruvate (PEP)

Reaction 6: Glyceraldehyde-3-P Dehydrogenase

G-3-P is oxidized to 1,3-BPG Oxidation of aldehyde to carboxylic acid is highly exergonic The overall reaction involves formation of carboxylic phosphoric anhydride and the reduction of NAD+ to NADH The overall reaction is slightly endergonic 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

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Rxn 6: Glyceraldehyde-3-P Dehydrogenase

Figure 18.14 A mechanism for the glyceraldehyde-3phosphate dehydrogenase reaction. Reaction of an enzyme sulfhydryl with G3P forms a thiohemiacetal, which loses a hydride to NAD+ to become a thioester. Phosphorolysis releases 1,3-bisphosphoglycerate.

G3P-DH
G3P-OH can be inactivated by reaction with iodoacetate, which reacts with and block the essential cysteine sulfhydryl

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.

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Reaction 7: Phosphoglycerate Kinase

ATP synthesis from a high-energy phosphate Phosphoglycerate kinase transfers a phosphoryl group from 1,3bisphosphoglycerate 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 Mg2+ is required (the true substrate is MgADP-) 2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction (Figure 18.15)

Reaction 7: Phosphoglycerate Kinase

Phosphoglycerate kinase transfers a phosphoryl group from 1,3bisphosphoglycerate to ADP to form ATP. This type of ATPsynthesizing reaction is referred to as a substrate-level phosphorylation

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Reaction 7: Phosphoglycerate Kinase

The open (a) and closed (b) forms of phosphoglycerate kinase. ATP (cyan), 3phosphoglycerate (purple), and Mg2+ (gold).

2,3-BPG is made by reactions that detour around the phosphoglycerate kinase reaction
2,3-bisphosphoglycerate is an important regulator of hemoglobin (see pages 505-506 of the text) 2,3-BPG is formed from 1,3-BPG by bisphosphoglycerate mutase 3-phosphoglycerate is then formed by 2,3bisphosphoglycerate phosphatase Most cells contain only a trace of 2,3-BPG, but erythrocytes typically contain 4-5 mM 2,3-BPG

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2,3-BPG is made by reactions that detour around the phosphoglycerate kinase reaction

Figure 18.15 Formation and decomposition of 2,3bisphosphglycerate.

2,3-BPG is made by reactions that detour around the phosphoglycerate kinase rxn

Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate. The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3phosphoglycerate.

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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 intermediates Zelda Rose (wife of Nobel laureate Irwin Rose) showed that a bit of 2,3-BPG is required to phosphorylate His Nomenclature note: a mutase catalyzes migration of a functional group within a substrate

Reaction 8: Phosphoglycerate Mutase

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Reaction 9: Enolase
Conversion of 2-Phosphoglycerate to PEP The enolase makes a high-energy phosphate in preparation for ATP synthesis in step 10 The overall G 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

Reaction 9: Enolase

The enolase reaction creates a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis.

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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 G indicating that this reaction is subject to regulation PK is allosterically activated by AMP, F-1,6-bisP PK is allosterically inhibited by ATP and acetyl-CoA Understand the keto-enol equilibrium of pyruvate; it is the key to understanding the pyruvate kinase reaction

Reaction 10: Pyruvate Kinase

The pyruvate kinase reaction converts PEP to pyruvate, driving synthesis of ATP. Note that two ATP are produced here, since two PEP are formed from one glucose. These two ATP are the payoff of glycolysis.

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Reaction 10: Pyruvate Kinase

Figure 18.19 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer, followed by an enol-keto tautomerization. The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis.

18.5 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: If O2 is available (aerobic conditions), NADH is oxidized in the electron transport pathway, making ATP in oxidative phosphorylation In anaerobic conditions, NADH is oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

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18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?

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.

18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?

Figure 18.21 (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction. Hibernating turtles, trapped beneath ice and lying in mud, become anoxic and convert glucose mainly to lactate. Their shells release minerals to buffer the lactate throughout the period of hibernation.

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18.5 What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis?

Pyruvate also represents energy - two possible fates:

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

The Warburg Effect and Cancer

Otto Warburg observed in 1924 that rapidly proliferating cancer cells metabolize glucose mainly to lactate, even when O2 is plentiful Lewis Cantley has suggested that this behavior arises because cells need more than ATP they must synthesize large amounts of nucleotides, amino acids, and lipids This requires lots of NADPH for biosynthesis as well as intermediates for building blocks Cancer cells divert large amounts of glucose to the pentose phosphate pathway to produce NADPH See page 597 for more details

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Otto Warburg

Otto Warburg, Nobel Prize in Physiology or Medicine, 1931 for his discovery of the nature and mode of action of the respiratory enzyme (cytochrome oxidase see Ch. 20)

The Warburg Effect and Cancer

Signaling proteins and pathways regulate the glycolytic pathway. Cancer cells route up to 90% of acquired glucose and glutamine into lactate and alanine, producing large amounts of NADPH

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18.6 How Do Cells Regulate Glycolysis?

The elegant evidence of regulation See Figure 18.22 Standard state G values are scattered, with both plus and minus values and no apparent pattern The plot of G values in cells is revealing: Most values near zero 3 of 10 reactions have large, negative G These 3 reactions with large negative G are sites of regulation (HK, PFK, PK) Regulation of these three reactions can turn glycolysis off and on

18.6 How Do Cells Regulate Glycolysis?

Figure 18.22 The free energies of the reactions of glycolysis under standardstate conditions.

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18.6 How Do Cells Regulate Glycolysis?

Figure 18.22 The free energies of the reactions of glycolysis under actual intracellular concentrations of metabolites in erythrocytes.

Tumor Diagnosis Using Positron Emission Tomography (PET)

Tumors show very high rates of glycolysis, as shown by Otto Warburg early in the 20th century This observation is the basis of tumor detection by positron emission tomography (PET) Metabolites labeled with 18F can be taken up by human cells (in the brain, for example) Decay of 18F results in positron emission Positron-electron collisions produce gamma rays Detection with gamma ray cameras provides 3D models of tumor extent and location 2-[18F]fluoro-2-deoxy-glucose, used for this purpose, is a substrate for hexokinase

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Tumor Diagnosis Using Positron Emission Tomography (PET)

2-[18F]fluoro-2-deoxyglucose is a substrate for hexokinase

Decay of 18F results in positron emission. Positron-electron collisions produce gamma rays

Detection with gamma ray cameras provides 3D models of tumor extent and location

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18.7 Are Substrates Other Than Glucose Used in Glycolysis?

Sugars other than glucose can be glycolytic substrates Fructose, mannose and galactose can all be used Fructose and mannose are routed into glycolysis by fairly conventional means. See Figure 18.23 Galactose is more interesting - the Leloir pathway "converts" galactose to glucose - sort of.... See Figure 18.24

18.7 Are Substrates Other Than Glucose Used in Glycolysis?

Figure 18.23 Mannose, galactose, fructose, and other simple metabolites can enter the glycolytic pathway.

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18.7 Are Substrates Other Than Glucose Used in Glycolysis?

Figure 18.24 Galactose metabolism via the Leloir pathway.

Galactose Enters Glycolysis Via the Leloir Pathway

Figure 18.25 The galactose-1-phosphate uridylyltransferase reaction involves a ping-pong kinetic mechanism.

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UDP-glucose pyrophosphorylase uses Gal-1-P, reducing galactose toxicity in adults Figure 18.26

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.

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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 dehydroxyacetone phosphate by the action of glycerol phosphate dehydrogenase.

Lactose From Mothers Milk to Yogurt and Lactose Intolerance

In placental mammals, lactose is synthesize only in the mammary gland, and then only during late pregnancy and lactation The synthesis is done by lactose synthase, a dimeric complex of galactosyl transferase and -lactalbumin Lactose breakdown in the intestines by lactase provides newborns with essential galactose Some humans are lactose intolerant, due to a particularly low level of lactase Lactic acid fermentation by certain bacteria is the basis for the production of yogurt

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Lactose From Mothers Milk to Yogurt and Lactose Intolerance

Breakdown of lactose to galactose and glucose by lactase.

Many Humans are Lactose Intolerant Due to a Low Level of Lactase

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18.8 How Do Cells Respond to Hypoxic Stress?

Glycolysis is an anaerobic pathway The tricarboxylic acid cycle is aerobic When oxygen is abundant, cells prefer to combine these pathways in aerobic metabolism When oxygen is limiting, cells adapt to carry out more glycolysis Hypoxia causes changes in gene expression that increases levels of glycolytic enzymes A trigger for this is a DNA-binding protein called hypoxia inducible factor (HIF) HIF is regulated at high oxygen levels by hydroxylase factorinhibiting HIF (FIH-1)

18.8 How Do Cells Respond to Hypoxic Stress?

HIF consists of two subunits: a ubiquitous HIF-1 subunit and a hypoxia-responsive HIF-1 subunit In response to hypoxia, inactivation of the prolyl hydroxylases allows
HIF-1 stabilization Dimerization with HIF-1 Binding of the dimer to the hypoxia-responsive element (HRE) of HIF target genes Activation of transcription of these genes

VHL is the von Hippel Lindau subunit of the ubiquitin E3 ligase that targets proteins for proteasome degradation

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18.8 How Do Cells Respond to Hypoxic Stress?

Figure 18.28

Chapter 19 The Tricarboxylic Acid Cycle

Dr Khairul Ansari

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Chapter 19
Thus times do shift, each thing his turn does hold; New things succeed, as former things grow old. Robert Herrick Hesperides (1648)

A time-lapse photograph of a ferris wheel at night. Aerobic cells use a metabolic wheel the TCA cycle to generate energy by acetyl-CoA oxidation.

How is pyruvate oxidized under aerobic conditions, and what is the chemical logic that dictates how this process occurs?

Essential Questions

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Outline
What is the chemical logic of the TCA cycle? How is pyruvate oxidatively decarboxylated to acetyl-CoA? How are two CO2 molecules produced from acetyl-CoA? How is oxaloacetate regenerated to complete the TCA cycle? What are the energetic consequences of the TCA cycle? Can the TCA cycle provide intermediates for biosynthesis? What are the anaplerotic, or filling up, reactions? How is the TCA cycle regulated? Can any organisms use acetate as their sole carbon source?

Oxidation of glucose to CO2 is a 24-electron oxidation

The electrons from glucose oxidation feed into the electron transport pathway, driving synthesis of ATP.

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Hans Krebs Showed That the Oxidation of Acetate is Accomplished by a Cycle

The TCA cycle is known as the Krebs cycle and also as the citric acid cycle Pyruvate from glycolysis is oxidatively decarboxylated to acetate Then acetate is degraded to CO2 in the TCA cycle Some ATP is produced More NADH is made NADH goes on to make more ATP in electron transport and oxidative phosphorylation

4 electrons are removed as NADH in glycolysis 2 NADH are produced during decarboxylation of two molecules of pyruvate For each acetyl-CoA oxidized in TCA cycle, 8 more electrons are removed as NADH (3) and FADH2(1) H3CCOO- + 2H20 + H+ 2CO2 + 8H In electron transport pathway these electrons combines with O2 to produce water 8H + 2O2 H2O Net reaction of TCA cycle and electron transport pathway is H3CCOO- + 2O2 + H+ 2CO2 + 2H2O

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Acetyl-CoA- entry point of new carbon into the TCA cycle Generally (in biological system) C-C occur cleavage between a and carbon Or between two carbons As acetate does not have a carbon neither of these cleavage are possible. Condensation of acetate with oxaloacetate facilitate cleavage

Figure 19.1 The Tricarboxylic Acid Cycle

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Figure 19.1 The Tricarboxylic Acid Cycle

Figure 19.1 The Tricarboxylic Acid Cycle

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19.1 What Is the Chemical Logic of the TCA Cycle?

The TCA cycle seems like a complicated way to oxidize acetate units to CO2 What are the possibilities (based on our knowledge of metabolism)? cleavage between Cs and to a carbonyl -cleavage of an -hydroxyketone These ways to cleave C-C bonds don't work for acetylCoA

Cleavage Between Carbon Atoms and to a Carbonyl

This type of cleavage occurs in the fructose bisphosphate aldolase reaction in glycolysis But it cant be used for acetate, which does not have a carbon

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-Cleavage of an -Hydroxyketone

This type of cleavage occurs in the transaldolase reaction (described in Chapter 22). But it would require hydroxylation of acetate, which is not a favorable or facile reaction for acetate Living things have evolved the clever chemistry of condensing acetate with oxaloacetate and then carrying out a -cleavage TCA combines this cleavage with oxidation to form CO2, regenerating oxaloacetate and capturing all the energy as NADH and ATP!

19.2 How Is Pyruvate Oxidatively Decarboxylated to AcetylCoA?

Pyruvate must enter the mitochondria to enter the TCA cycle Oxidative decarboxylation of pyruvate is catalyzed by the pyruvate dehydrogenase complex Pyruvate dehydrogenase is a noncovalent assembly of three enzymes Five coenzymes are required

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Figure 19.4 The Reaction Mechanism of the Pyruvate Dehydrogenase Complex

Decarboxylation of pyruvate yields hydroxyethyl-TPP. Transfer to lipoic acid is followed by formation of acetyl-CoA.

19.3 How Are Two CO2 Molecules Produced from AcetylCoA?

The citrate synthase synthase reaction initiates the TCA cycle Citrate synthase is classic CoA chemistry C of the acetyl group in acetyl-CoA is acidic and can be deprotonated to form a carbanion This carbanion is a strong nucleophile that can attack the -carbonyl of oxaloacetate, yielding citryl-CoA Thioester hydrolysis then produces citrate NADH & succinyl-CoA are allosteric inhibitors Note (Table 19.1) that citrate synthase has a large negative G and is a site of regulation

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The citrate synthase reaction initiates the TCA cycle

Figure 19.6 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nucleophilic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis. NADH is an allosteric inhibitor of citrate synthase

The citrate synthase synthase reaction initiates the TCA cycle

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The citrate synthase synthase reaction initiates the TCA cycle

Figure 19.7 Citrate synthase in mammals is a dimer of 49-kD subunits. In the monomer shown here, citrate (blue) and CoA (red) bind to the active site, which lies in a cleft between two domains and is surrounded mainly by -helical segments.

Citrate Is Isomerized by Aconitase to Form Isocitrate

Citrate is a poor substrate for oxidation So aconitase isomerizes citrate to yield isocitrate which has a secondary -OH, which can be oxidized Note the stereochemistry of the reaction: aconitase removes the pro-R H of the pro-R arm of citrate Aconitase uses an iron-sulfur cluster - see Figure 19.8

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Citrate Is Isomerized by Aconitase to Form Isocitrate

Figure 19.8 The aconitase reaction converts citrate to cis-aconitate and then to isocitrate. Aconitase is stereospecific and removes the pro-R hydrogen from the pro-R arm of citrate.

Citrate Is Isomerized by Aconitase to Form Isocitrate

Figure 19.8 The active site of aconitase. The iron-sulfur cluster (pink) is coordinated by cysteines (orange) and isocitrate (purple).

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Fluoroacetate Blocks the TCA Cycle

Fluoroacetate is an extremely poisonous agent that blocks the TCA cycle in vivo, although it has no apparent effect on any of the isolated TCA cycle enzymes. The action of aconitase has been traced to aconitase Aconitase is inhibited by fluorocitrate, which is formed from fluoracetate in two steps, as shown here.

Isocitrate Dehydrogenase Catalyzes the First Oxidative Decarboxylation in the Cycle

Oxidative decarboxylation of isocitrate yields -ketoglutarate This is classic NAD+ chemistry (hydride removal) followed by a decarboxylation Isocitrate dehydrogenase is a link to the electron transport pathway because it makes NADH The mechanism is typical of NAD+-dependent enzymes (see Figure 19.10) The reaction involves (first) oxidation of the C-2 alcohol of isocitrate to form oxalosuccinate, then a -decarboxylation reaction that expels the central carboxyl group as CO2

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Isocitrate Dehydrogenase Catalyzes the First Oxidative Decarboxylation in the Cycle

Figure 19.10 The isocitrate dehydrogenase reaction. Hydride removal by NAD+ is followed by a -decarboxylation reaction that expels the central carboxyl group as CO2

-Ketoglutarate Dehydrogenase Catalyzes the Second Oxidative Decarboxylation of the TCA Cycle
A second oxidative decarboxylation This enzyme is nearly identical to pyruvate dehydrogenase structurally and mechanistically Five coenzymes used - TPP, CoASH, lipoic acid, NAD+, and FAD You know the mechanism if you remember pyruvate dehydrogenase

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-Ketoglutarate Dehydrogenase Catalyzes the Second Oxidative Decarboxylation of the TCA Cycle

Like pyruvate dehydrogenase, -ketoglutarate dehydrogenase is a multienzyme complex consisting of -ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and dihydrolipoyl dehydrogenase. The complex uses five different coenzymes.

19.4 How Is Oxaloacetate Regenerated to Complete the TCA Cycle?

Succinyl-CoA Synthetase Catalyzes a Substrate-Level Phosphorylation A nucleoside triphosphate is made This is possible because succinyl-CoA is a high-energy intermediate Its hydrolysis (the hydrolysis of a CoA ester) drives the phosphorylation of GDP to produce GTP The mechanism (Figure 19.11) involves a phosphohistidine

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19.4 How Is Oxaloacetate Regenerated to Complete the TCA Cycle?

Hydrolysis of succinyl-CoA (a CoA ester) drives the phosphorylation of GDP to produce GTP

Completion of the TCA Cycle Oxidation of Succinate to Oxaloacetate

This process involves a series of three reactions that will be seen five times throughout the text These reactions include: Oxidation of a single bond to a double bond Hydration across the double bond Oxidation of the resulting alcohol to a ketone This trio of reactions will be seen again in: Fatty acid breakdown and synthesis Amino acid breakdown and synthesis

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Succinate Dehydrogenase Is FAD-Dependent

An FAD-dependent oxidation of a single bond to a double bond The mechanism involves hydride removal by FAD and a deprotonation This enzyme is actually part of the electron transport pathway in the inner mitochondrial membrane The electrons transferred from succinate to FAD (to form FADH2) are passed directly to ubiquinone (UQ) in the electron transport pathway

The Succinate Dehydrogenase Reaction

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Fumarase Catalyzes the trans-Hydration of Fumarate to Form L-Malate

Hydration occurs across the newly formed double bond Hydration involves trans-addition of the elements of water across the double bond Possible mechanisms are shown in Figure 19.13 The actual mechanism is not known for certain

The Fumarase Reaction

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Possible Mechanisms For the Fumarase Reaction

Figure 19.13

Malate Dehydrogenase Completes the Cycle by Oxidizing Malate to Oxaloacetate

An NAD+-dependent oxidation The carbon that gets oxidized is the one that received the -OH in the previous reaction This reaction is energetically expensive: Go' = +30 kJ/mol Thus the concentration of oxaloacetate in the mitochondrial matrix is quite low However, the malate dehydrogenase reaction is pulled forward by the favorable citrate synthase reaction

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The Malate Dehydrogenase Reaction

Steric Preferences in NAD+-Dependent Dehydrogenases

The dehydrogenases that require nicotinamide coenzymes are stereospecific, and they transfer hydride to either the pro-R or the pro-S positions selectively What accounts for this specificity? The enzymes involved are asymmetric structures. The nicotinamide coenzyme (and substrate) fit into the active site in only one way The hydride transfers can only be accomplished to or from one side or the other of the NAD+ molecule Dehydrogenases (and other enzymes too) are also stereospecific with respect to the substrates as well

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19.6 What Are the Energetic Consequences of the TCA Cycle?

One acetate through the cycle produces two CO2, one ATP, four reduced coenzymes The equations on page 625-626 summarize the stoichiometry and energetics of the TCA cycle A useful exercise would be to try to derive these equations (or at least justify each term) Try also to account for the overall energetics of this pathway The TCA cycle is exergonic, with a net G' for one pass around the cycle of -40 kJ/mol The combination of glycolysis and TCA produce 12 reduced coenzymes, which can eventually produce as much as 34 molecules of ATP Net energy reaction combining Glycolysis and TCA cycle together Glucose + 2 H2O + 10 NAD+ + 2[FAD] + 4 ADP + 4Pi6 CO2 + 10 NADH + 10 H+ + 2 [FADH2] + 4ATP

The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle
The carbonyl C of acetyl-CoA becomes CO2 only in the second turn of the cycle (following entry of acetyl-CoA ) The methyl C of acetyl-CoA survives two cycles completely, but half of what's left exits the cycle on each turn after that The C-C bond cleaved in a given TCA cycle actually entered as an acetate in the previous turn Thus the oxidative decarboxylations that cleave this bond are just a disguised acetate C-C cleavage and oxidation

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The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle

Figure 19.15 The fate of the carbon atoms of acetate in successive TCA cycles.

The Carbon Atoms of Acetyl-CoA Have Different Fates in the TCA Cycle

Figure 19.15 The fate of the carbon atoms of acetate in successive TCA cycles.

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19.7 Can the TCA Cycle Provide Intermediates for Biosynthesis?

The TCA cycle provides several of these -Ketoglutarate is transaminated to make glutamate, which can be used to make purine nucleotides, as well as Arg and Pro Succinyl-CoA can be used to make porphyrins Fumarate and oxaloacetate can be used to make several amino acids and also pyrimidine nucleotides Note that mitochondrial citrate can be exported to be a cytoplasmic source of acetyl-CoA and oxaloacetate

The TCA Cycle Can Provide Intermediates For Biosynthesis

Figure 19.16 The TCA cycle provides intermediates for biosynthesis. Amino acids are highlighted in orange. All 20 common amino acids can be made from metabolites derived from the TCA cycle.

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19.8 What Are the Anaplerotic, or Filling Up, Reactions?

Pyruvate carboxylase - converts pyruvate to oxaloacetate This is the most important anaplerotic reaction PEP carboxylase - converts PEP to oxaloacetate Malic enzyme converts pyruvate into malate PEP carboxykinase - could have been an anaplerotic reaction, but it goes the wrong way CO2 binds weakly to PEP carboxykinase, but oxaloacetate binds tightly, so the reaction goes spontaneously in the opposite direction.

19.8 What Are the Anaplerotic, or Filling Up, Reactions?

Figure 19.17 Pyruvate carboxylase (shown here), and also phosphoenolpyruvate (PEP) carboxylase, and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates.

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19.8 What Are the Anaplerotic, or Filling Up, Reactions?

Figure 19.17 Pyruvate carboxylase , phosphoenolpyruvate (PEP) carboxylase (shown here), and malic enzyme catalyze anaplerotic reactions, replenishing TCA cycle intermediates.

19.8 What Are the Anaplerotic, or Filling Up, Reactions?

Figure 19.17 Pyruvate carboxylase, and also phosphoenolpyruvate (PEP) carboxylase and malic enzyme (shown here) catalyze anaplerotic reactions, replenishing TCA cycle intermediates.

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Is PEP Carboxykinase an Anaplerotic Reaction?

This reaction *might* be an anaplerotic reaction, except for the fact that CO2 binds weakly to PEP carboxykinase, and oxaloacetate binds very tightly. As a result, the enzyme favors formation of PEP from oxaloacetate. Thus the reaction operates in the wrong direction to be an anaplerotic reaction.

Anaplerosis Plays a Critical Role in Insulin Secretion

The pancreas releases insulin in response to an increase of blood glucose What cellular processes mediate this response? It was long accepted that ATP produced by catabolism activated K+ channels in the plasma membrane of -cells in the pancreas However, recent research has shown that anaplerotic enzymes feed alternative pathways that produce cytosolic signal molecules that also support insulin secretion

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Anaplerosis Plays a Critical Role in Insulin Secretion

Pancreatic -cells maintain high levels of the anaplerotic enzyme pyruvate carboxylase In these cells, half the pyruvate from glycolysis is converted by pyruvate carboxylase to oxaloacetate, much of which is converted to malate and exported to the cytosol, as part of a pyruvate/malate cycle NADPH and other metabolites generated from this cycle act as intracellular messengers that appear to be as significant as ATP in provoking insulin release Anaplerosis also plays a role in peripheral tissues: In insulinresistant individuals, exercise increases anaplerotic activity, increases fatty acid oxidation, and restores insulin sensitivity

Anaplerosis Plays a Critical Role in Insulin Secretion

The pyruvate/malate cycle in pancreatic -cells. Oxaloacetate produced by high levels of pyruvate carboxylase is converted to malate, then exported to the cytosol.

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The Reductive TCA Cycle

The TCA cycle running backward could assimilate CO2 This may have been the first metabolic pathway This reductive TCA cycle occurs in certain extant archaea and bacteria, where it serves all their carbon needs Energy to drive it? Maybe reaction of FeS with H2S to form FeS2 (iron pyrite) Iron pyrite, which was plentiful in ancient times, is an ancient version of iron-sulfur clusters found in many enzymes today!

The Reductive TCA Cycle

A reductive, reversed TCA cycle

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19.9 How Is the TCA Cycle Regulated?

As in TCA, 3 reactions are the key sites Citrate synthase - ATP, NADH and succinyl-CoA inhibit Isocitrate dehydrogenase - ATP inhibits, ADP and NAD+ activate -Ketoglutarate dehydrogenase - NADH and succinyl-CoA inhibit, AMP activates Also note pyruvate dehydrogenase: ATP, NADH, acetyl-CoA inhibit NAD+, CoA activate

Figure 19.18 Regulation of the TCA cycle.

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Pyruvate Dehydrogenase is Regulated by Phosphorylation/Dephosphorylation

Figure 19.19 Regulation of the pyruvate dehydrogenase reaction. Phosphorylation inactivates; Dephosphorylation activates.

19.10 Can Any Organisms Use Acetate as Their Sole Carbon Source?
The Glyoxylate Cycle Acetate-based growth - net synthesis of carbohydrates and other intermediates from acetate - is not possible with TCA The glyoxylate cycle offers a solution for plants and some bacteria and algae The CO2-evolving steps are bypassed and an extra acetate is utilized Isocitrate lyase and malate synthase are the short-circuiting enzymes

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19.10 Can Any Organisms Use Acetate as Their Sole Carbon Source?

Figure 19.20 The glyoxylate cycle. The first two steps are identical to TCA reactions. Isocitrate lyase and malate synthase short-circuit the TCA cycle, forming malate and succinate from isocitrate and another acetyl-CoA.

More on the Glyoxylate Cycle

Isocitrate lyase produces glyoxylate and succinate Malate synthase does a Claisen condensation of acetyl-CoA and the aldehyde group of glyoxylate - classic CoA chemistry! The glyoxylate cycle helps plants grow in the dark! Seeds are a rich source of acetate (from fatty acids). Until the nascent plant sees the sun (and begins photosynthesis), it can grow using the glyoxylate cycle

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Isocitrate Lyase Short-Circuits the TCA Cycle by Producing Glyoxylate and Succinate

Figure 19.21 The isocitrate lyase reaction. Isocitrate lyase catalyzes an aldol cleavage and is similar to the aldolase reaction in glycolysis.

Glyoxysomes Must Borrow Three Reactions from Mitochondria

Glyoxysomes do not contain all the enzymes needed to run the glyoxylate cycle Succinate dehydrogenase, fumarase, and malate dehydrogenase are absent Glyoxysomes borrow these three reactions from mitochondria, so that they can convert succinate to oxaloacetate

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Glyoxysomes Must Borrow Three Reactions from Mitochondria

Figure 19.20 Glyoxysomes lack three of the enzymes needed to run the glyoxylate cycle. Succinate dehydrogenase, fumarase, and malate dehydrogenase are all borrowed from mitochondria.

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