Glycolysis

UNIT II:

Intermediary Metabolism

Overview• In cells reactions rarely occur in isolation, but rather

organized into multi-step sequences called pathways, such as glycolysis

• In a pathway, product of one reaction serves as substrate of subsequent reaction

• Different pathways can also intersect, forming an integrated and purposeful network of chemical reactions. These are collectively called metabolism, which is the sum of all chemical changes occurring in a cell, a tissue or the body.

• Most pathways can be classified as either catabolic (degradative) or anabolic (synthetic).

• Catabolic reactions break down complex molecules e.g., proteins, polysacch’s & lipids, to a few simple molecules e.g., CO2, NH3 & H2O

• Anabolic pathway form complex end products from simple precursors e.g., synthesis of the polysacch, glycogen, from glucose

Figure 8.1Glycolysis, an example of a metabolic pathway.

A. Metabolic map

- It is convenient to investigate metabolism by examining its component pathways

- Each pathway is multienzyme sequences, and each enz in turn may exhibit important catalytic or regulatory feature

- Metabolic map is useful in tracing connections b/w pathways, visualizing purposeful movement of metabolic intermediates, and picturing the effect on flow of intermediates if a pathway is blocked e.g., by a drug or inherited deficiency of an enz

Figure 8.2. Important reactions of intermediary metabolism. Several important pathways tobe discussed in later chapters are highlighted. Curved reaction arrows ( )indicate forward and reverse reactions that are catalyzed by different enzymes.The straight arrows ( ) indicate forward and reverse reactions that arecatalyzed by the same enzyme. Key: Blue text = intermediates of carbohydratemetabolism; brown text = intermediates of lipid metabolism; green text =intermediates of protein metabolism.

B. Catabolic pathways- Catabolic reactions serve to capture chemical energy in

the form of ATP from degradation of energy-rich fuel molecules

- Catabolism also allows molecules in diet (or nutrient molecules stored in cells) to be converted to building blocks needed for synthesis of complex molecules

- Energy generation by degradation of complex molecules occurs in 3 stages:

1. Hydrolysis of complex molecules: complex molecules are broken down into their component building blocks. E.g., proteins aa’s, polysacch’s monosacch’s & triglycerides free fatty acids and glycerol

2. Conversion of building blocks to simple intermediates: diverse building blocks further degraded to acetyl CoA and a few other simple molecules.

Some energy is captured as ATP, but amount is small compared with that produced during 3rd stage

3. Oxidation of acetyl CoA: TCA cycle is the final common pathway in oxidation of fuel molecules such as acetyl CoA.

Large amounts of ATP are generated as e’s flow from NADH & FADH2 to O2 via oxphos

Figure 8.3. Three stages of catabolism.

C. Anabolic pathway- Anabolic reactions combine small molecules, e.g., aa’s

to form complex molecules, such as proteins- Anabolic reactions require energy, which is generally

provided by breakdown of ATP to ADP + Pi- Anabolic reactions often involve chemical reductions in

which reducing power is most frequently provided by e-donor NADPH

- Note that catabolism is a convergent process i.e., a wide variety of molecules are transformed into a few common end products

- By contrast, anabolism is a divergent process i.e., a few biosynthetic precursors form a wide variety of polymeric or complex products

Figure 8.4. Comparison of catabolic and anabolic pathways.

II. Regulation of metabolism- Pathways of metabolism must be coordinated so

that production of energy or synthesis of end products meets needs of cell

- Individual cells do not function in isolation but, rather, are part of a community of interacting tissues

- Thus, a sophisticated communication system has evolved to coordinate functions of the body

- Regulatory signals that inform an individual cell of the metabolic state of the body as a whole include hormones, neurotransmitters, and the availability of nutrients. These, in turn, influence signals generated within the cell

A. Signals from within the cell (intracellular)- Rate of a metabolic pathway can respond to

regulatory signals that arise from within cell- E.g., rate of a pathway may be influenced by

availability of substrates, product inhibition, or alterations in levels of allosteric activators or inhibitors

- These intracellular signals typically elicit rapid responses, and are important for moment-to-moment regulation of metabolism

B. Communication between cells (intercellular)- Ability to respond to extracellular signals is essential

for survival & development of all organisms- Signaling b/w cells provides for long-range integration

of metabolism, & usually results in a response that is slower than is seen with signals that originates within the cell

- Communication b/w cells can be mediated by surface-to-surface contact &, in some tissues, by formation of gap junctions, allowing direct communication b/w cytoplasms of adjacent cells

- However, for energy metabolism, the most important route of communication is chemical signaling e.g., by blood-borne hormones or by neurotransmitters

C. Second messenger systems- Hormones or neurotransmitters can be thought of as

signals, & a receptor as a signal detector. Each component serves as a link in the communication b/w extracellular events & chemical changes within the cell

- Many receptors signal their recognition of a bound ligand by initiating a series of reactions that ultimately result in a specific intracellular response

- “second messenger” molecules, so named as they intervene b/w original messenger (neurotransmitter or hormone) & the ultimate effect on cell, are part of the cascade of events that translates hormone or neurotransmitter binding into a cellular response

- Two of the most widely recognized 2nd messenger systems are the calcium/phosphatidylinositol system & adenylyl cyclase system, which is particularly important in regulating pathways of intermediary metabolism

Figure 8.5Some commonly used mechanisms for transmission of regulatory signals between cells.

D. Adenylyl cyclase- Recognition of a chemical signal by some memb

receptors, such as β- & α2-adrenergic receptors, triggers either an increase or a decrease in the activity of adenylyl cyclase.

- This is a memb-bound enz that converts ATP to 3`,5`-adenosine monophosphate (a.k.a cyclic AMP or cAMP)

- Chemical signals are most often hormones or neurotransmitters, each of which binds to a unique type of memb receptor

- Therefore, tissues that respond to more than one chemical signal must have several different receptors, each of which can be linked to adenylyl cyclase

Note: certain toxins, as that produced by Vibrio cholera, can also activate the adenylyl cyclase cascade, with potentially disastrous consequences

- These receptors are characterized by an extracellular ligand-binding region, 7 transmembrane helices, & an intracellular domain that interacts with G-proteins

Figure 8.6Structure of a typical membrane receptor.

1. GTP-dependent regulatory proteins:- Effect of activated occupied receptor on 2nd messenger

formation is not direct, it is mediated by specialized trimeric proteins in the CM.

- These, referred to as G-proteins because they bind guanosine nucleotides (GTP & GDP), form a link in the chain of communication b/w receptor & adenylyl cyclase

- The inactive form of G-protein binds to GDP- The activated receptor interacts with G-proteins,

triggering an exchange of GTP for GDP.- The timeric G-proteins then dissociates into an α

subunit & a βγ dimer.- The GTP-bound form of the α subunit moves from the

receptor to adenylyl cyclase, which is thereby activated

- Many molecules of active G-protein are formed by one activated receptor

Note: ability of a hormone or neurotransmitter to stimulate or inhibit adenylyl cyclase depends on the type of G-protein that is linked to the receptor. One family of G-proteins, designated Gs, is specific for stimulation of adenylyl cyclase; another family, Gi inhibition of the enz.

- Actions of G-protein-GTP complex are short-lived because the G-protein has an inherent GTPase activity rapid hydrolysis of GTP to GDP inactivation of G-protein

Figure 8.7. The recognition of chemical signals by certain membrane receptors triggers an increase (or, less often, a decrease) in the activity of adenylyl cyclase.

2. Protein kinases:- Next link in cAMP 2nd-messenger system is activation by

cAMP of a family of enz’s = cAMP-dependent protein kinases, e.g., protein kinase A.

- cAMP activates protein kinase A by binding to its two regulatory subunits, causing release of active catalytic subunits.

- The active subunits catalyze the transfer of P from ATP to specific ser or thr residues of protein substrates

- Phosphorylated proteins may act directly on cell’s ion channels, or may become activated or inhibited enz’s

- Protein kinase A can also phosphorylate specific proteins that bind to promoter regions of DNA increased expression of specific genes

- Note: not all protein kinases respond to cAMP; there are several types of protein kinases that are not cAMP-dependent, e.g., protein kinase C

Figure 8.8. Actions of cAMP.

3. Dephosphorylation of proteins:- P groups added to proteins by protein kinases are

removed by protein phosphatases, enz’s that hydrolytically cleave phosphate esters

- This ensures that changes in enzymatic activity induced by protein phosphorylation are not permanent

4. Hydrolysis of cAMP:- cAMP is rapidly hydrolyzed to 5`-AMP by cAMP

phosphodiesterase, one of a family of enz’s that cleave cyclic 3`,5`-phosphodiester bond

- 5`-AMP is not an intracellular signaling molecule. Thus, effects of neurotransmitter or hormone-mediated increases of cAMP are rapidly terminated if the extracellular signal is removed

Note: phosphodiesterase is inhibited by methylxanthine derivatives, such as theophylline & caffeine

III. Overview of glycolysis

- Glycolytic pathway is employed by all tissues for the breakdown of glucose to provide energy (in form of ATP) and intermediates for other metabolic pathways

- Glycolysis is at the hub of CHO metabolism because virtually all sugars, whether arising from diet or from catabolic reactions in the body, can ultimately be converted to glucose

- Pyruvate is the end product of glycolysis in cells with mitochondria & an adequate supply of oxygen

- This series of 10 reactions is called aerobic glycolysis because oxygen is required to reoxidize NADH formed during oxidation of glyceraldehyde-3-P

- Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of TCA cycle

- Alternatively, glucose can be converted to pyruvate, which is reduced by NADH lactate. This is called anaerobic glycolysis because it can occur without participation of oxygen.

- Anaerobic glycolysis allows the continued production of ATP in tissues that lack mitochondria (e.g., RBCs) or in cells deprived of sufficient oxygen

Figure 8.9. A. Glycolysis shown as one of the essential pathways of energy metabolism. B. Reactions of aerobic glycolysis. C. Reactions of anaerobic glycolysis.

IV. Transport of glucose into cells

- Glucose cannot diffuse directly into cells, but enters by one of two transport mechanisms: a Na+-independent, facilitated diffusion transport system, or a Na+-monosacch co-transporter system

A. Na+-independent facilitated diffusion transport- This system is mediated by a family of at least 14

glucose transporters in CMs. They are = GLUT-1 to GLUT-14 (glucose transporter isoform 1-14)

- These transporters exist in membrane in two conformational states. Extracellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the CM

Figure 8.10Schematic representation of the facilitated transport of glucose through a cell membrane.

1. Tissue specificity of GLUT gene expression:

- Glucose transporters display a tissue-specific pattern of expression e.g., GLUT-3 is the primary glucose transporter in neurons. GLUT-1 is abundant in erythrocytes & brain, but is low in adult muscle, whereas GLUT-4 is abundant in adipose tissue & skeletal muscle

Note: number of GLUT-4 transporters active in these tissues is increased by insulin. The other GLUT isoforms also have tissue-specific distributions

2. Specialized functions of GLUT isoforms:

- In facilitated diffusion, glucose movement follows a conc. gradient, i.e., from a high gluc conc. to a lower one. E.g., GLUT-1, -3, -4 are primarily involved in gluc uptake from blood

- In contrast, GLUT-2 which is found in liver, kidney, and β-cells of pancreas, can either transport gluc into these cells when blood gluc levels are high, or transport gluc from cells to blood when blood glucose levels are low (e.g., during fasting)

- GLUT-5 is unusual in that it is the primary transporter for fructose (instead of gluc) in the small intestine & testes

- GLUT-7 which is expressed in liver & other gluconeogenic tissues, mediates glucose flux across endoplasmic reticulum memb.

B. Na+-monosaccharide cotransporter system

- This is an energy-requiring process that transports glucose “against” a conc. gradient i.e., from low gluc conc’s outside the cell to higher conc’s within cell

- This system is a carrier-mediated process in which movement of glucose is coupled to the conc gradient of Na+, which is transported into cell at the same time.

- This type of transport occurs in epithelial cells of intestine, renal tubules, and choroid plexus

V. Reactions of glycolysis

• Conversion of gluc to pyruvate occurs in 2 stages. The 1st

five reactions correspond to an energy investment phase in which phosphorylated forms of intermediates are synthesized at the expense of ATP

• The subsequent reactions of glycolysis constitute an energy generation phase in which a net of 2 molecules of ATP are formed by substrate level phosphorylation per gluc molecule metabolized

Note: 2 molecules of NADH are formed when pyruvate is produced (aerobic glycolysis), whereas NADH is converted to NAD+ when lactate is produced (anaerobic glycolysis)

Figure 8.11Two phases of aerobic glycolysis.

A. Phosphorylation of glucose- Phosphorylated sugar molecules do not readily

penetrate CMs, because no specific transmemb carriers for these cpds, & they are too polar to diffuse through the CM

- The irreversible phosphorylation of gluc, therefore, effectively traps the sugar as cytosolic G-6-P, thus committing it to further metabolism in the cell

- Mammals have several isozymes of the enz hexokinase that catalyze the phosphrylation of gluc to G-6-P

Figure 8.12Energy investment phase: phosphorylation of glucose.

1. Hexokinase:- In most tissues, phosphorylation of gluc is catalyzed by

hexokinase, one of 3 regulatory enz’s of glycolysis (phosphofructokinase I & pyruvate kinase are the other two)

- Hexokinase has broad substrate specificity and is able to phosphorylate several hexoses in addition to gluc.

- Hexokinase is inhibited by the reaction product, G-6-P, which accumulates when further metabolism of this hexose-P is reduced

- Hexokinase has a low Km (&, therefore, a high affinity) for gluc. This permits efficient phosphorylation & subsequent metabolism of gluc even when tissue conc’s of gluc are low

- Hexokinase, however, has a low Vmax for gluc &, therefore, cannot sequester (trap) cellular phosphate in the form of phosphorylated hexoses, or phosphorylate more sugars than the cell can use

2. Glucokinase:- In liver parenchymal cells & islet cells of the

pancreas, glucokinase (also called hexokinase D, or type IV) is the predominant enz responsible for the phosphorylation of gluc

- In β-cells, glucokinase functions as gluc sensor, determining the threshold for insulin secretion

- In liver, the enz facilitates gluc phosphorylation during hyperglycemia

Note: despite its misleading name “glucokinase” the sugar specificity of the enz is similar to that of other hexokinase isozymes

a. Kinetics:- Glucokinase differs from hexokinase in several

important properties- E.g., it has much higher Km, requiring a higher gluc conc for

half-saturation. Thus glucokinase functions only when intracellular conc of gluc in hepatocyte is elevated, e.g., during the brief period following consumption of a CHO-rich meal, when high levels of gluc are delivered to the liver via the portal vein

- Glucokinase has a high Vmax, allowing the liver to effectively remove the flood of gluc delivered by the portal blood. This prevents large amounts of gluc from entering systemic circulation following a CHO-rich meal, & thus minimizes hyperglycemia during the absorptive period

Note: GLUT-2 insures that blood gluc equilibrates rapidly across the memb of the hepatocyte

Figure 8.13Effect of glucose concentration on the rate of phosphorylation catalyzed byhexokinase and glucokinase.

b. Regulation by fructose 6-phosphate and glucose:

- Glucokinase activity is not allosterically inhibited by G-6-P as are other hexokinases, but rather is indirectly inhibited by F-6-P (which is in equil. with G-6-P), & is stimulated indirectly by glucose via the following mechanism:- A glucokinase regulatory protein exists in the nucleus of

hepatocytes.- In presence of F-6-P, glucokinase is translocated into nucleus &

binds tightly to regulatory protein, thus rendering enz inactive- When gluc levels in the blood (& also in the hepatocyte, as a

result of GLUT-2) increase, the gluc causes the release of glucokinase from the regulatory protein, & the enz enters the cytosol where it phosphorylates gluc to G-6-P

- As free gluc levels fall, F-6-P causes glucokinase to translocate back into nucleus & bind to regulatory protein, thus inhibiting enz’s activity

Figure 8.14Regulation of glucokinase activity by glucokinase regulatory protein.

c. Regulation by insulin:

- Glucokinase activity in hepatocytes is also increased by insulin

- as blood gluc levels rise following a meal, β-cells of pancreas are stimulated to release insulin into portal circulation

Note: ~ ½ of the newly secreted insulin is extracted by liver during the 1st pass through that organ. Therefore, liver is exposed to twice as much insulin as is found in systemic circulation

- insulin also promotes transcription of glucokinase gene, resulting in an increase in liver enz protein &, therefore, of total glucokinase activity.

Note: the absence of insulin in patients with diabetes causes a deficiency in hepatic glucokinase. This contributes to an inability of patient to efficiently decrease blood glucose levels

B. Isomerization of glucose-6-phosphate- Isomerization of G-6-P to F-6-P is catalyzed by

phosphoglucose isomerase. Reaction is readily reversible & is not a rate-limiting or regulated step

Figure 8.15Isomerization of glucose 6-phosphate to fructose 6-phosphate.

C. Phosphorylation of fructose 6-phosphate- The irreversible phosphorylation reaction catalyzed

by phosphofructokinase-1 (PFK-1) is the most important control point & the rate-limiting step of glycolysis

- PFK-1 is controlled by available conc’s of the substrates ATP & F-6-P, & by the following regulatory substances:

1. Regulation by energy levels within the cell: - PFK-1 is inhibited allosterically by elevated levels of

ATP, which act as an “energy-rich” signal indicating an abundance of high-energy cpds.

- Elevated levels of citrate, an intermediate in the TCA cycle, also inhibit PFK-1. Conversely, PFK-1 is activated allosterically by high conc’s of AMP, which signal that the cells’ energy stores are depleted

2. Regulation by fructose 2,6-bisphosphate- F-2,6-bisP is the most potent activator of PFK-1.

This cpd also acts as an inhibitor of fructose 1,6-bisphosphatase

- The reciprocal actions of F-2,6-bisP on glycolysis & gluconeogenesis ensure that both pathways are not fully active at the same time

Note: this would result in a “futile cycle” in which glucose would be converted to pyruvate followed by re-synthesis of glucose from pyruvate

- F-2,6-bisP is formed PFK-2, an enz different than PFK-1. F-2,6-bisP is converted back to F-6-P by fructose bisphosphatase-2

Note: the kinase & phosphatase activities are different domains of one bifunctional polyp molecule

Figure 8.16Energy investment phase (continued): Conversion of fructose 6-phosphate to triose phosphates.

a. During the well-fed state:- Decreased levels of glucagon & elevated

levels of insulin, such as occur following a CHO-rich meal, cause an increase in F-2,6-bisP and thus the rate of glycolysis in the liver.

- F-2,6-bisP, therefore acts as an intracellular signal, indicating that glucose is abundant

b. During starvation:- Elevated levels of glucagon & low levels of

insulin, such as occur during fasting, decrease the intracellular conc of hepatic F-2,6-bisP. This results in a decrease in the overall rate of glycolysis and an increase in gluconeogenesis

Figure 8.17. Effect of elevated insulin concentration on the intracellular concentration of fructose 2,6-bisphosphate in liver.PFK-2 = phosphofructokinase-2; FBP-2 = Fructose bisphospate phosphatase-2.

D. Cleavage of fructose 1,6-bisphosphate- Aldolase A cleaves F-1,6-bisP to dihydroxyacetone-

P (DHAP) & glyceraldehyde-3-P (GA-3P). The reaction is reversible & not regulated

Note: Aldolase B in the liver and kidney also cleaves F-1,6-bisP, & functions in metabolism of dietary fructose

E. Isomerization of dihydroxyacetone phosphate- Triose phosphate isomerase interconverts DHAP

& GA-3P. DHAP must be isomerized to GA-3P for further metabolism by glycolytic pathway

- This isomerization results in net production of 2 GA-3P from cleavage products of F-1,6-bisP

Figure 8.16Energy investment phase (continued): Conversion of fructose 6-phosphate to triose phosphates.

F. Oxidation of glyceraldehyde 3-phosphate- Conversion of GA-3P to 1,3-bisP glycerate by

GA-3P dehydrogenase is 1st redox reaction of glycolysis

Note: because there is only a limited amount of NAD+ in the cell, NADH formed by this reaction must be reoxidized to NAD+ for glycolysis to continue.

- Two major mechanisms for oxidizing NADH are:

1) NADH-linked conversion of pyruvate to lactate

2) oxidation of NADH via respiratory chain

1. Synthesis of 1,3-bisphosphoglycerate (1,3-BPG):

- Oxidation of aldehyde group of GA-3P to a carboxyl group is coupled to attachment of Pi to the carboxyl group

- The high-energy P group at C-1 of 1,3-BPG conserves much of the free energy produced by oxidation of GA-3P

- The energy of this high-energy P drives synthesis of ATP in the next reaction of glycolysis

2. Mechanism of arsenic poisoning:- Toxicity of arsenic is explained primarily by inhibition

of enz’s such as pyruvate dehydrogenase, which require lipoic acid as a cofactor. However, pentevalent arsenic (arsenate) also prevents net ATP & NADH production by glycolysis, without inhibiting the pathway itself.

- The poison does so by competing with Pi as a substrate for GA-3P dehydrogenase, forming a complex that spontaneously hydrolyzes to form 3-P-glycerate

- By bypassing synthesis and dephosphorylation of 1,3-BPG, the cell is deprived of energy usually obtained from glycolytic pathway

3. Synthesis of 2,3-BPG in RBCs:

- Some 1,3-BPG is converted to 2,3-BPG by bisphospho-glycerate mutase

- 2,3-BPG, which is found in only trace amounts in most cells, is present at high conc in RBCs

- 2,3 BFG is hydrolyzed by a phosphatase to 3-phosphoglycerate, which is also an intermediate in glycolysis

- In RBCs, glycolysis is modified by inclusion of these “shunt” reactions

Figure 8.18. Energy-generating phase: conversion of glyceraldehyde 3-phosphate to pyruvate.

G. Synthesis of 3-phosphoglycerate producing ATP- When 1,3-BPG is converted to 3-phosphoglycerate,

the high-energy P group of 1,3-BPG is used to synthesize ATP from ADP

- This reaction is catalyzed by phosphoglycerate kinase, which, unlike most other kinases, is physiologically reversible

- Because 2 molecules of 1,3-BPG are formed from each gluc molecule, this kinase reaction replaces the 2 ATP molecules consumed by earlier formation of G-6-P & F-1,6-BP

Note: this is an example of substrate level phospho., in which production of a high-energy P is coupled directly to oxidation of substrate, instead of resulting from oxidative phosph. via ETC.

H. Shift of phosphate group from carbon 3 to carbon 2

- Shift of P group from C-2 to C-3 of phosphoglycerate by phosphoglycerte mutase is freely reversible

I. Dehydration of 2-phosphoglycerate

- Dehydration of 2-phosphoglycerate by enolase distributes the energy within 2-phosphoglycerate formation of PEP, which contains a high-energy enol phosphate. The reaction is reversible despite the high-energy nature of the product

J. Formation of pyruvate producing ATP- Conversion of PEP to pyruvate is catalyzed by

pyruvate kinase. The 3rd irreversible reaction of glycolysis.

- The equil of pyruvate kinase reaction favors formation of ATP

Note: this is another example of substrate level phosphorylation

1. Feed-forward regulation:- In liver, pyruvate kinase is activated by F-1,6-BP, the

product of PFK reaction. This feed-forward regulation has the effect of linking the 2 kinase activities: increased PFK activity elevated levels of F-1,6-BP activates pyruvate kinase

2. Covalent modulation of pyruvate kinase:- Phosphorylation by a cAMP-dependent protein kinase

leads to inactivation of pyruvate kinase in the liver- When blood gluc levels are low, elevated glucagon

increases intracellular level of cAMP causes phosphorylation & inactivation of pyruvate kinase. Therefore, PEP is unable to continue in glycolysis, but instead enters gluconeogenesis pathway.

- This, in part, explains the observed inhibition of hepatic glycolysis & stimulation of gluconeogenesis by glucagon.

- Dephosphorylation of pyruvate kinase by a phosphoprotein phosphatase results in reactivation of enz

Figure 8.19Covalent modification of pyruvate kinase results in inactivation ofenzyme

3. Pyruvate kinase deficiency:

- Normal, mature RBC lacks mitochondria & is therefore, completely dependent on glyolysis for production of ATP.

- ATP is required to meet metabolic needs of RBC, & also to fuel the pumps necessary for maintenance of bi-concave, flexible shape of the cell, which allows it to squeeze through narrow capillaries

- Anemia observed in glycolytic enz deficiencies is a consequence of reduced rate of glycolysis, leading to decreased ATP production.

- Resulting alterations in RBC memb lead to changes in shape of cell, and ultimately, to phagocytosis by cells of reticuloendothelial system, particularly macrophages of spleen

- Premature death & lysis of RBC hemolytic anemia

- Among patients exhibiting genetic defects of glycolytic enz’s, ~ 95% show a deficiency in pyruvate kinase, & 4% exhibit phosphoglucose isomerase deficiency

- Pyruvate kinase (PK) deficiency is the 2nd most common cause (after G-6PD deficiency) of enzymatic related hemolytic anemia

- PK deficiency is restricted to RBCs, & produces mild to severe chronic hemolytic anemia (RBC destruction), with the severe form requiring regular cell transfusions

- Severity of disease depends both on degree of enz deficiency (generally 5-25% of normal levels), & on extent to which individual’s RBCs compensate by synthesizing increased levels of 2,3-BPG

- Almost all individuals with PK deficiency have a mutant enz that shows abnormal properties- most often altered kinetics

Figure 8.20Alterations observed with various mutant forms of pyruvate kinase.

K. Reduction of pyruvate to lactate- Lactate, formed by action of lactate dehydrogenase, is

final product of anaerobic glycolysis in euk cells. Formation of lactate is major fate of pyruvate in RBCs, lens & cornea of the eye, kidney medulla, testes, & leukocytes

1. Lactate formation in muscle:- In exercising skeletal muscle, NADH production (by

glyceraldehyde 3-P dehydrogenase & by the three NAD+-linked dehydrogenases of TCA cycle) exceeds oxidative capacity of respiratory chain

- This results in elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate. Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in intracellular pH, potentially resulting in cramps.

- Much of this lactate eventually diffuse into bloodstream, & can be used by liver to make glucose

Figure 8.21. Interconversion of pyruvate and lactate.

2. Lactate consumption:- The direction of lactate dehydrogenase reaction depends

on relative intracellular conc’s of pyruvate & lactate & on ratio of NADH/NAD+ in the cell. E.g., in liver & heart, ratio of NADH/NAD+ is lower than in exercising muscle. These tissues oxidize lactate (obtained from blood) to pyruvate

- In liver, pyruvate is either converted to glucose by gluconeogenesis or oxidized in the TCA cycle

- Heart muscle exclusively oxidizes lactate to CO2 & H2O via citric acid cycle

3. Lactic acidosis:- Elevated conc’s of lactate in plasma termed

lactic acidosis occur when there is a collapse of the circulatory system, e.g., in MI, pulmonary embolism, & uncontrolled hemorrhage, or when an individual is in shock

- Failure to bring adequate amounts of oxygen to the tissues impaired oxidative phosphorylation & decreased ATP synthesis

- To survive, cells use anaerobic glycolysis as a backup system for generating ATP, producing lactic acid as the end-product

Note: production of even meager amounts of ATP may be life-saving during the period required to re-establish adequate blood flow to the tissues

- The excess oxygen required to recover from a period when the availability of oxygen has been inadequate is termed “oxygen debt”.

- The oxygen debt is often related to patient morbidity or mortality.

- In many clinical situations, measuring the blood levels of lactic acid provides for the rapid, early detection of oxygen debt in patients.

- E.g., blood lactic acid levels can be used to measure the presence & severity of shock, & to monitor the patients’ recovery

L. Energy yield from glycolysis- Despite production of some ATP during

glycolysis, end-products, pyruvate or lactate, still contain most of energy originally contained in gluc. The TCA cycle is required to release that energy completely.

1. Anaerobic glycolysis:- Two molecules of ATP are generated for

each molecule of gluc converted to 2 molecules of lactate. There is no net production or consumption of NADH.

- Anaerobic glycolysis, although releasing only a small fraction of energy contained in gluc molecule, is a valuable source of energy under several conditions, including:

1. when oxygen supply is limited, as in muscle during intensive exercise2. for tissues with few or no mitoch., such as the medulla of the kidney, mature RBCs, leukocytes, & cells of the lens, cornea & testes

2. Aerobic glycolysis:- Direct formation & consumption of ATP is the

same as in anerobic glycolysis i.e., a net gain of 2 ATP

- Two molecules of NADH are also produced per molecule of gluc

- Ongoing aerobic glycolysis requires the oxidation of most of this NADH by ETC, producing ~ 3 ATP for each NADH entering the chain (depending on the shuttle system)

Figure 8.22. Summary of anaerobic glycolysis. Reactions involving the production or consumption of ATP or NADH are indicated. The irreversible reactions of glycolysis are shown with thick arrows. DHAP = dihydroxyacetone phosphate

VI. Hormonal regulation of glycolysis- Regulation of glycolysis by allosteric activation or

inhibition, or phospho/dephospho of rate-limiting enz’s, is short-term i.e., they influence gluc consumption over periods of minutes or hours

- Superimposed on these moment-to-moment effects are slower, often more profound, hormonal influences on amount enz protein synthesized. These effects can result in 10x to 20x fold increases in enz activity that typically occur over hours to days

- Although current focus is on glycolysis, reciprocal changes occur in the rate-limiting enz’s of gluconeogenesis

- Regular consumption of meals rich in CHO or administration of insulin initiates an increase in the amount of glucokinase, PFK, & PK in liver

- These changes reflect an increase in gene transcription, resulting in increased enz synthesis.

- High activity of these 3 enz’s favors conversion of gluc to pyruvate, a characteristic of the well-fed state

- Conversely, gene transcription and synthesis glucokinase, PFK, & PK are decreased when plasma glucagon is high & insulin is low, e.g., as seen in fasting or diabetes

Figure 8.23Effect of insulin and glucagon on the synthesis of key enzymes of glycolysis in liver.

VII. Alternate fates of pyruvateA. Oxidative decarboxylation of pyruvate- Oxidative decarboxylation of pyruvate by pyruvate

dehydrogenase complex is an important pathway in tissues with high oxidative capacity, e.g., cardiac muscle.

- Pyruvate dehydrogenase irreversibly converts pyruvate, the end product of glycolysis, into acetyl CoA, a a major fuel for TCA & the building block for fatty acid synthesis

B. Carboxylation of pyruvate to oxaloacetate- Carboxylation of pyruvate to OAA by pyruvate caroxylase

is a biotin-dependent reaction. This reaction is important because it replenishes TCA cycle intermediates, & provides substrate for gluconeogenesis

Figure 8.24Summary of the metabolicfates of pyruvate.

C. Reduction of pyruvate to ethanol (microorganisms)

- Conversion of pyruvate to ethanol occurs by two reactions (Fig. 8-24)

- Decarboxylation of pyruvate by pyruvate decarboxylase occurs in yeast & certain m/o’s, but not in humans. The enz requires thiamine pyrophosphate as a coenz, & catalyzes a reaction similar to that described for pyruvate dehydrogenase

Summary

• Most pathways can be classified as either catabolic (degrade complex molecules to a few simple products) or anabolic (synthesize complex end products from simple precursors)

• Catabolic reactions also capture chemical energy in form of ATP from degradation of energy-rich molecules

• Anabolic reactions require energy, which is generally provided by breakdown of ATP

• Rate of a metabolic pathway can respond to regulatory signals, e.g., allosteric activators or inhibitors, that arise within the cell

• Signaling b/w cells provides for integration of metabolism• The most important route of this communication is

chemical signaling b/w cells, e.g., by hormones or neurotransmitters

• Second messenger molecules convey the intent of a chemical signal (hormone or neurotransmitter) to appropriate intracellular responders.

• Adenylyl cyclase is a memb-bound enz that syntheizes cAMP in response to chemical signals, e.g., the hormones glucagon & epinephrine

• Following binding to a hormone to its cell-surface receptor, a GTP-dependent regulatory protein (G-protein) is activated that, in turn, activates adenylyl cyclase

• The cAMP activates a protein kinase, which phosphorylates a cadre of enz’s, causing their activation or deacivation/ ion channels regulation / gene expression

• Phospho is reversed by protein phosphatases

• Aerobic glycolysis, in which pyruvate is the end product, occurs in cells with mitoch & an adequate supply of oxygen

• Anaerobic glycolysis, in which lactic acid is the end product, occurs in cells that lack mitoch, or in cells deprived of sufficient oxygen

• Gluc is transported across memb’s by one of at least 14 glucose transporter isoforms (GLUTs). GLUT-1 is abundant in RBCs & brain, GLUT-4 (which is insulin-dependent) is found in muscle & adipose tissue, & GLUT-2 is found in liver & the β-cells of the pancreas

• Conversion of gluc to pyruvate (glycolysis) occurs in 2 stages: an energy investment phase in which phospho intermediates are synthesized at expense of ATP, & an energy generation phase, in which ATP is produced

• In energy investment phase, gluc is phosphorylated by hexokinase (found in most tissues) or glucokinase (a hexokinase found in liver cells & the β cells of pancreas)

• Hexokinase has a high affinity (low Km) & a small Vmax for glucose, & is inhibited by G-6-P

• Glucokinase has a large Km & a large Vmax for glucose. It is indirectly inhibited by F6P & activated by gluc, & the transcription of the glucokinase gene is enhanced by insulin.

• G6P is isomerized to F6P, which is phosphorylated to F-1,6-BP by PFK. This enz is allosterically inhibited by ATP & citrate, & activated by AMP.

• F-2,6-BP, whose synthesis is activated by insulin, is the most potent allosteric activator of this enz. A total of 2 ATP are used during this phase of glycolysis.

• F-1,6-BP is cleaved 2 trioses further metabolized by glycolytic pathway pyruvate

• During these reactions, 4 ATP & 2 NADH are produced from ADP & NAD+.

• Final step in pyruvate synthesis fro PEP is catalyzed by PK. This enz is allosterically activated by F-1,6-BP, & hormonally activated by insulin & inhibited by glucagon via cAMP pathway.

• PK deficiency accounts for 95% of all inherited defects in glycolytic enz’s. it is restricted to RBCs, & causes mild to severe chronic hemolytic anemia.

• In anaerobic glycolysis, NADH is reoxidized to NAD+ by conversion of pyruvate to lactic acid. This occurs in cells, e.g., RBCs, that have few or no mitoch., & in tissues , such as exercising muscle, where production of NADH exceeds oxidative capacity of respiratory chain

• Elevated conc’s of lactate in plasma (lactic acidosis) occur when there is collapse of the circulatory system, or when an individual in shock

• Pyruvate can be:

1. Oxidatively decarboxylated by pyruvate dehydrogenase, producing acetyl CoA

2. Carboxylated to OAA by pyruvate carobxylase

3. Reduced by m/o’s to ethanol by pyruvate decarboxylase

Figure 8.25Key concept map for glycolysis.