glycolysis - marquette university -

Glycolysis

Objectives: I. Digestion of carbohydrates

A. Location(s) of carbohydrate digestion B. Enzymes involved

1. Enzyme source C. Stereochemical nature of the glycosidic linkages necessary for digestion.

1. What is lactose intolerance? 2. What enzyme is faulty / absent?

II. Glycolysis - The pathway A. Describe why Glycolysis is (usually) the first metabolic pathway examined. B. Distinguish its two major segments. C. Describe the central role of glucose in carbohydrate catabolism. D. Describe the glycolysis pathway E. Describe the entry of the other hexoses into glycolysis.

1. The different, tissue specific, modes of fructose entry 2. What is Galactosemia?

a) What enzyme or enzymes are faulty? F. Given a step or the steps of glycolysis, indicate the type of reaction that has occurred and

possible enzyme involved. G. Which step(s) demonstrate substrate level phosphorylation?

III. Pyruvate A. Describe the aerobic and anaerobic fates of pyruvate.

1. Generation of lactate and regeneration of NAD by lactate dehydrogenase. 2. How are the Km’s of the lactate dehydrogenase isoenzymes different? 3. How do the different Km’s affect the reaction equilibrium / direction of reaction?

IV. Glycolysis - The Control A. What types of reactions are catalyzed by the rate controlling enzymes? B. Describe the allosteric modulators and their effects on the allosteric enzymes that control

glycolysis. C. Describe the hormonal regulation of glycolysis.

V. Ask yourself “What If Questions”; e.g., What happens to the process of glycolysis if glyceraldehyde-3-phosphate dehydrogenase is inhibited?

Background

We have reached the study of metabolic interconversions. You will find in the coming lectures that many biomolecules and enzyme names are introduced. If a systematic approach is taken, these pathways will not be overwhelming. It is essential to learn the names of the key enzymes, since in many cases they will be encountered again in connection with other pathways. Keep in mind that chemical structures of metabolites prompt enzyme names, and the name of the enzymes reflect the substrate specificity and the type of reaction catalyzed. With a confident grasp of terminology, you will be prepared to enjoy the chemical elegance of metabolism. However, it is important not to lose sight of the important concepts and general strategies of metabolism while learning the details. The names of particular enzymes might fade from memory after the

©Kevin R. Siebenlist, 20181

final exam, but I hope you will retain an understanding of the patterns and purposes behind the interconversions of metabolites in cells. Paraphrased from Moran et. al., pg. 15-3

Carbohydrate Digestion

CARBOHYDRATE DIGESTION begins in the mouth with Salivary α-Amylase. This enzyme is an endo-glucanase. It catalyzes the hydrolysis of starch and glycogen at random internal locations liberating maltose, maltotriose, glucose, dextrins, and random length oligosaccharides. DEXTRINS are disaccharides or trisaccharides containing the glucose α1 → 6 glucose branch point of amylopectin and/or glycogen. Carbohydrate digestion stops in the stomach because the low pH of the stomach secretions denature salivary α-amylase. In the small intestine, Pancreatic α-Amylase completes the hydrolysis of starch and glycogen into maltose, glucose, and dextrins. Enzymes expressed on the outer surface of the epithelial cells lining the small intestine complete the digestion of disaccharides.

1. Dextrinase cleaves the glucose α1 → 6 glucose bond dextrin liberating glucose. 2. Maltase hydrolyzes maltose into two molecules of glucose. 3. Sucrase releases glucose and fructose from sucrose. 4. Lactase hydrolyzes lactose into galactose and glucose.

The surface of small intestine epithelial cells also contain hydrolases that cleave the glycosidic bonds of the heteropolysaccharides that are attached to proteins and/or lipids. The amount of “sugar” present in the heteropolysaccharides is small compared to the “sugar” obtained from the homopolysaccharides and disaccharides (starch, glycogen, lactose & sucrose).

Individuals who suffer from LACTOSE INTOLERANCE lack the enzyme Lactase. Several genetically distinct populations of the human animal stop synthesizing Lactase once they are weaned and/or as they approach adulthood. Without this enzyme any lactose in the diet passes through the small intestine unchanged. In the large intestine the anaerobic bacterial flora metabolize the undigested lactose and generate many noxious gaseous waste products.

Glycolysis

GLYCOLYSIS from the Greek; “GLYKYS-, sweet” and “LYSIS, to loosen, to split”. Glycolysis is the preparatory catabolic pathway that begins the breakdown and oxidation of glucose and other hexoses for energy. As originally characterized, glycolysis is a sequence of 10 enzyme catalyzed reactions during which glucose is converted into two molecules of the α-keto acid, pyruvate. The chemical energy released during this metabolic interconversion is trapped in two molecules of ATP and two molecules of NADH.

The net balanced reaction of glycolysis is:

Glucose + 2 ADP + 2 PO4–3 + 2 NAD+ → 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

Glycolysis is studied first for several reasons.


1. Glycolysis was the first pathway completely elucidated and it is the most extensively studied pathway. It was elucidated in the first half of the 20th century by Otto Warburg, Gustav Embden, and Otto Meyerhof. The pathway is sometimes called the Embden-Meyerhof pathway.

2. Glycolysis is a ubiquitous pathway. All living cells contain some form of the glycolytic pathway. This implies that glycolysis is a very old pathway, one of the first to evolve, and it suggests that the breakdown of sugars for energy is important for life processes.

3. It is the only metabolic pathway in mammalian cells that can generate energy in the form of ATP in the absence of molecular oxygen. It is an anaerobic pathway.

4. It is the only energy generating catabolic pathway that occurs in the cytoplasm of eukaryotic cells. The other energy producing pathways occur in the mitochondria.

5. Glycolysis exhibits many of the underlying themes of metabolism.

The glycolytic pathway can be divided into two phases. The first phase is called the Preparatory Phase, the Energy Expenditure Phase, or the Hexose Phase. During this part of the pathway the hexose is prepared for cleavage and then cleaved into two three carbon sugars. Energy, obtain from ATP, is expended during this phase. The second phase is the Energy Generation Phase, the Energy Recovery Phase, or the Triose Phase. During this part of the pathway the three carbon sugars derived from glucose are converted to the α-keto acid, pyruvate. Energy is liberated during this phase and is stored in ATP and as high energy electrons carried by NADH.

Glycolysis - The Pathway

In the first step of the pathway a phosphate group is transferred from ATP to glucose to form glucose-6-phosphate and ADP. This irreversible reaction is catalyzed by Hexokinase I, Hexokinase II, Hexokinase III or Hexokinase IV (Glucokinase). These enzymes are ISOENZYMES. Resting blood glucose concentration is about 5 × 10–3 M (5 mM). The Km of hexokinase I, II, III is in the range of 5 × 10–4 M to 5 × 10–6 M (0.005 to 0.5 mM). Hexokinase I, II & III is always saturated with substrate. Hexokinase IV (Glucokinase) is found in the liver and to a lesser extent in the pancreas. The Km of glucokinase is about 10 mM. Glucokinase is seldom saturated with substrate under resting conditions. It functions primarily after a meal when blood glucose levels are very high. Glucokinase allows the liver to phosphorylate, capture, and store large amounts of glucose thereby rapidly lowering blood glucose concentrations. Glucokinase activity in the pancreas plays a role in controlling insulin and glucagon release. Transfer of phosphate to glucose serves several purposes. It traps glucose in the cell. Since glucose-6-phosphate can not bind to the passive glucose transporters, it can not be transported out of the cell. Conversion into glucose-6-phosphate keeps the glucose levels in the cell lower than the plasma so


O

OH OH

OH

OH

CH2OH

ATP

ADP

Hexokinaseor

Glucokinase

O

OH OH

OH

OH

CH2OP

O

O

O

Glucose-6-phosphateIsomerase

orPhosphohexoseIsomerase

CH2

OH

CH2OH

OH

OHO

OP

O

O

O

Glucose

Glucose-6-phosphate

Fructose-6-phosphate

(1)

(2)

glucose is always transported into the cell. The addition of phosphate also activates and labels glucose for glycolysis or one of the other pathways involving glucose.

In the second step glucose-6-phosphate is isomerized into fructose-6-phosphate. This reaction is catalyzed by Glucose-6-phosphate isomerase (also called Phosphoglucoisomerase or Phosphohexose isomerase). This reaction is freely reversible and favors the formation of glucose-6-phosphate.

During the third step of the pathway a second phosphate group is transferred to the hexose. ATP is the phosphate donor and the product is fructose-1,6-bisphosphate and ADP. The reaction is catalyzed Phosphofructokinase-1. Addition of the second phosphate labels both ends of the molecule with a highly charged phosphate group and prepares it for cleavage into two three carbon sugars. If both ends were not phosphorylated, upon cleavage one fragment could diffuse or be transported from the cell and be lost. This reaction is irreversible.

Fructose-1,6-bisphosphate is now split into a pair triose phosphates (4). Carbon 1, 2, & 3 becomes dihydroxyacetone phosphate and carbon 4, 5, & 6 becomes glyceraldehyde-3-phosphate. This reaction is an aldol cleavage (the reverse of an aldol condensation). This reversible reaction is catalyzed by Fructose-1,6-bisphosphate aldolase or just Aldolase.

The fifth step is an isomerization


Fructose-1,6-bisphosphate

TriosephosphateIsomerase

CH2

OH

H2C

OH

OHO

OP

O

O

O

O P O

O

O

C

HC

H2C

OH

OH

O P

O

O

O

H2C

C

CH2OP

O

O

O

OH

O

Dihydroxyacetonephosphate

Glyceraldehyde-3-phosphate

PO4–3

NAD+

NADH

Glyceraldehyde-3-phosphateDehydrogenase

(Fructose-1,6-bisphosphate)Aldolase

C

HC

H2CO P

O

O

O

OH

O OP

O

O

O1,3-Bisphosphoglycerate

CH2

OH

CH2OH

OH

OHO

OP

O

O

O Fructose-6-phosphate

ATP

ADP

Phosphofructokinase-1(3)

(4)

(5)

(6)

reaction. Dihydroxyacetone phosphate is reversibly isomerized to glyceraldehyde-3-phosphate. This reaction is catalyzed by Triosephosphate isomerase. Converting dihydroxyacetone phosphate to glyceraldehyde-3-phosphate simplifies the remaining steps of the pathway and minimizes the number of enzymes necessary to complete the pathway. The cell is able to utilize both halves of the glucose (hexose) molecule effectively. The equilibrium position of this reaction favors the formation of dihydroxyacetone phosphate. However, utilization of glyceraldehyde-3-phosphate in the subsequent reactions of glycolysis pulls this reaction toward glyceraldehyde-3-phosphate formation. This reaction marks the end of the first phase of the glycolytic pathway.

During the second phase of the pathway the triose phosphate intermediates are converted from low energy states to high energy phosphorylated forms by a series of bond rearrangements. The high energy phosphate is then passed from the high energy intermediate to ADP forming ATP and a low energy intermediate. Reactions that synthesize ATP by the transfer of a high energy phosphate from a “substrate” molecule to ADP are called SUBSTRATE LEVEL PHOSPHORYLATIONS. This sequence of events; conversion from low energy compound to high energy compound followed by high energy phosphate transfer, is a common motif in intermediary metabolism and it occurs twice in the last five reactions of glycolysis. Remember, each glucose molecule that enters glycolysis yields two trioses and each of the last five reactions of the pathway must occur twice if the entire glucose molecule is to be used for energy generation.

In the sixth step of the pathway, the first step of the second half, glyceraldehyde-3-phosphate is oxidized. The aldehyde group on carbon one is oxidized to a carboxylic acid group. Some of the energy released by the oxidation is stored as a pair of electrons (H–) on NADH. The remainder is used to form a high energy mixed phosphoanhydride bond between a PO4–3 from the cytoplasm and the


C

HC

H2COH

O

O O

P

O

O

O

C

C

CH2

O

O O

P

O

O

O

C

HC

H2CO P

O

O

O

OH

O O

H2O

PhosphoglycerateMutase

Enolase

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

C

HC

H2CO P

O

O

O

OH

O O

ADP

ATP

P

O

O

O1,3-Bisphosphoglycerate

(7)

(8)

(9)

PhosphoglycerateKinase

new carboxylate group. The ultimate product is 1,3-bisphosphoglycerate. This reversible reaction is catalyzed by Glyceraldehyde-3-phosphate dehydrogenase. Arsenate [AsO4–3] (a poison) is an inhibitor of glycolysis. Arsenate substitutes for phosphate in the reaction catalyzed by Glyceraldehyde-3-phosphate dehydrogenase and is added to the intermediate during the reaction. The high energy product, 1-arseno-3-phosphoglycerate is very unstable and spontaneously breaks down to 3-phosphoglycerate and AsO4–3 without the generation of ATP.

The high energy phosphate group on carbon one of 1,3-bisphosphoglycerate is now passed to ADP forming ATP (7). The other product is 3-phosphoglycerate. This reaction is an example of substrate level phosphorylation. Phosphoglycerate kinase catalyzes this reversible reaction. At this point the cell has broken even. Two ATPs were used during the first phase of glycolysis and two ATPs are generated at this point when both halves of the glucose molecule pass through these reactions.

Low energy 3-phosphoglycerate is isomerized into 2-phosphoglycerate by moving the phosphate from the primary hydroxyl group on C-3 to the secondary –OH group on C-2 (8). 2-Phosphoglycerate is also a low energy compound. This reversible reaction is catalyzed by Phosphoglycerate mutase. {mutase - a subclass of isomerases specific for moving phosphates} This reaction prepares the molecule for the dehydration reaction that will occur in the next step. Primary alcohols are easier for the cell to dehydrate than secondary alcohols.

In this step, 2-phosphoglycerate is dehydrated to form phosphoenolpyruvate (9). ENOLS contain a carbon-carbon double bond (ene) with a hydroxyl group (ol) bonded to one of the carbons of the double bond. Enols are unstable, high energy compounds and adding a phosphate in ester linkage to the hydroxyl group makes it more high energy. Phosphoenolpyruvate (PEP) is a high energy intermediate. This reversible reaction is catalyzed by Enolase (also called Phosphopyruvate hydratase or 2-phosphoglycerate dehydratase). Enolase is competitively inhibited by fluoride ion.

During the last step of glycolysis (10) the high energy phosphate of phosphoenolpyruvate is transferred to ADP forming ATP. This is the second example of a substrate level phosphorylation. This irreversible reaction is catalyzed by Pyruvate kinase. Since each glucose molecule yields two phosphoenolpyruvate, 2 ATP are generated along with two pyruvate at this step.

Entry of Other Hexoses into Carbohydrate Metabolism

The reactions that convert the other hexoses, Mannose, Galactose, and Fructose, into intermediates that can enter glycolysis, for the most part converts these hexoses into forms that can enter many of the pathways of “mainstream” carbohydrate metabolism. Keep in mind that once these hexoses are converted into an intermediate of glycolysis, catabolism by the glycolytic pathway is not their only fate, they can be used by


C

C

CH2

O

O O

P

O

O

O

ADP

ATP

PyruvateKinase

Phosphoenolpyruvate

C

C

CH3

O

O O

PYRUVATE

(10)

any of the other pathways of carbohydrate metabolism.

Mannose

Mannose is phosphorylated by the action of Hexokinase to form mannose-6-phosphate (see below). Mannose-6-phosphate is then reversibly isomerized into fructose-6-phosphate by the action of Phosphomannose Isomerase. The fructose-6-phosphate can now enter carbohydrate metabolism.

Fructose

O

OH OH

OH

OH

CH2OH

O

OH OH

OH

OH

CH2OP

OO

O

CH2

OH

CH2OH

OH

OHO

OP

O

O

O

CH2

OH

H2C

OH

OHO

OP

O

O

O

O P O

O

O

ATP

ADP

ATP

ADP

Hexokinaseor

Glucokinase

Glucose-6-phosphate Isomeraseor

Phosphohexose Isomerase

Phosphofructokinase I

Glucose

Glucose-6-phosphate



HOCH2

OH

CH2OH

OH

OHO

ATPADP

Hexokinase

ATP

ADP

Hexokinase

Mannose-6-phosphate

Mannose

Fructose

Phosphomannose Isomerase

O

OH

OH

OHOH

H2CO P

O

O

O

O

OH

OH

OHOH

CH2OH


The entry of Fructose into “mainstream” carbohydrate metabolism depends upon the tissue in which the reactions are occurring. In most tissues (e.g., muscle, kidney, adipose tissue, etc.) fructose enters metabolism after it phosphorylated by the action of Hexokinase to form fructose-6-phosphate (see above).

In the liver, and only in the liver, fructose is phosphorylated to fructose-1-phosphate by the action of Fructokinase. Hexokinase IV (Glucokinase) does not use fructose as a substrate. Fructose-1-phosphate is


Dihydroxyacetonephosphate

Glyceraldehyde-3-phosphate

PO4–3

NAD+

NADH

Glyceraldehyde-3-phosphateDehydrogenase

(Fructose-1-phosphate)Aldolase

HOCH2

OH

CH2OH

OH

OH

O

C

HC

H2COH

OH

OH

ATP

ADP

C

HC

H2CO

OH

OH

PO

O

O

CH2

C

CH2

O

HO

O

PO

O

O

TriosephosphateIsomerase

Fructose(in the Liver)

Fructokinase

ATP

ADP

Glyceraldehyde

Triosekinase

CH2OH

OH

H2C

OH

OH

O

O P O

O

O


then cleaved into dihydroxyacetone phosphate and glyceraldehyde by the action of Fructose-1-phosphate Aldolase. The dihydroxyacetone phosphate enters “mainstream” carbohydrate metabolism. The glyceraldehyde is phosphorylated by the action of Triose Kinase and the resulting glyceraldehyde-3-phosphate enters metabolism.

Galactose

Of the three hexoses, the entry of Galactose into carbohydrate metabolism is the most complex (see pathway

O

OH OH

OH

OH

CH2OP

O

O

O

Glucose-6-phosphate

Galactokinase

Galactose-1-phosphateuridylyltransferase

UDP-Glucose-4-epimaerase

Phosphoglucomutase

Galactose

Galactose-1-phosphate

Glucose-1-phosphate

UDP-Glucose

UDP-Galactose

Glucose-6-phosphate

Glucose-1-phosphateUTP

P2O7

Phosphoglucomutase

UDP-GlucosePyrophosphorylase

O

OH O

OH

OH

CH2OH

P

O

O

O

OH O

OH

OH

OH

CH2OH

OH O

O

OH

OH

CH2OH

P

O

O

O

O

OH O

OH

OH

CH2OHNH

O

ON

O

OHOH

CH2OPO

O

O

P

O

O

NH

O

ON

O

OHOH

OPO

O

O

P

OH O

O

OH

OH

CH2OH

O

O

ATP

ADP


above). Galactose is first phosphorylated by the action of Galactokinase to form galactose-1-phosphate. The Galactose-1-phosphate then takes part in a transferase reaction with UDP-glucose. UDP-glucose is the nucleotide uridine diphosphate with the anomeric hydroxyl group of glucose in ester linkage to the β phosphate. UDP-glucose is the activated form of glucose used for biosynthetic and epimerase reactions. Galactose-1-phosphate Uridylyltransferase catalyzes the transferase reaction between UDP-glucose and galactose-1-phosphate. The products are glucose-1-phosphate and UDP-galactose. Glucose-1-phosphate is reversibly isomerized to glucose-6-phosphate by the action of Phosphoglucomutase and the glucose-6-phosphate enters metabolism. Several reactions have occurred, but the galactose is still galactose. The UDP-galactose that is formed is reversibly converted to UDP-glucose by the action of UDP-Glucose-4-Epimerase. The net result of these four reactions is the conversion of galactose to glucose-6-phosphate. GALACTOSEMIA is a genetic disease due to disordered galactose metabolism. In the most common form of galactosemia, the enzyme Galactose-1-phosphate Uridylyltransferase is defective. Other forms of galactosemia result when either Galactokinase or UDP-Glucose-4-Epimerase are genetically defective.

Lactate Dehydrogenase

The cell has a finite concentration of NAD+. In order for glycolysis to continue the NADH formed at the glyceraldehyde-3-phosphate dehydrogenase step must be oxidized (back) to NAD+. Under normal (aerobic) conditions the NADH formed during glycolysis is oxidized to NAD+ by the second of the two final common pathways. In this pathway NADH passes its electrons, its hydride ion in a series of reaction steps to oxygen; the NADH is oxidized to NAD+ and the oxygen is reduced to H2O. Most mammalian tissues are always well oxygenated and the electrons generated by glycolysis (NADH) are passed to the second final common pathway. All tissues can function anaerobically for short periods of time, three to five minutes before irreversible damage occurs. However, rapidly contracting skeletal muscle can function for a more prolonged period of time under anaerobic conditions. For glycolysis to continue under anaerobic conditions in all tissues and especially in the muscle cell there must be an alternative method to regenerate the NAD+. If the NAD+ is not regenerated, glycolysis would stop when all of the cytoplasmic NAD+ has been reduced to NADH. Glycolysis is the primary source of ATP for the anaerobically contracting muscle and if it stops muscle contraction stops. Under anaerobic conditions the NAD+ is regenerated by the action of the enzyme Lactate dehydrogenase (LDH). This enzyme catalyzes the reversible reduction of pyruvate to lactate. The NAD+ reenters glycolysis at the glyceraldehyde-3-phosphate dehydrogenase step and the glycolytic pathway can continue to operate.

Lactate is a dead end intermediate. Before it can be utilized by the cell it must be oxidized to pyruvate. When the skeletal muscle cell is adequately oxygenated, lactate is converted to pyruvate using NAD+ as the electron (hydride) acceptor. Lactate dehydrogenase (LDH) catalyzes this back reaction.

Lactate dehydrogenase (LDH) is the classic example of an isoenzyme. Isoenzymes catalyze the same reaction but they have different primary structures. However, since they catalyze the same reaction the 2°, 3°, and 4° structures are similar, the higher order structures


C

HC

CH3

OH

O O

Lactate

C

C

CH3

O

O O

Pyruvate

LactateDehydrogenase

NAD+

NADH

NAD+

NADH

show a high degree of homology. Isoenzymes are often tissue specific, different tissues contain different isoenzymes to perform slightly different, tissue specific functions.

Human Lactate dehydrogenase is a multimeric enzyme composed of a total of four subunits. Lactate dehydrogenase is not an allosteric enzyme - all allosteric enzymes are multimeric, not all multimeric enzymes are allosteric. The human cell contains genes for two different Lactate dehydrogenase subunits; the Heart or H subunit and the Muscle or M subunit. When these subunits are combined into the active tetrameric enzyme, five different combinations of subunits, five different isoenzymes are possible; H4, H3M, H2M2, HM3, and M4. The H4 form is heart specific, the H4 and H3M forms are richest in kidney, red blood cells contain H3M, the HM3 and M4 forms are richest in skeletal muscle, and the liver contains only M4.

Skeletal muscle is the only tissue that can tolerate anaerobic conditions for extended periods of time. The M subunit of Lactate dehydrogenase has a low Km, high affinity for pyruvate and a high Km, low affinity for lactate. With this arrangement of Km’s and subunit compositions (HM3 and M4), the skeletal muscle Lactate dehydrogenase catalyzes the formation of lactate from pyruvate very efficiently at low pyruvate concentrations. The reverse reaction - lactate to pyruvate - requires high concentrations of lactate. High concentrations of lactate are present in the skeletal muscle cell upon re-oxygenation, after the prolonged exercise is over. Rapidly contracting skeletal muscle or skeletal muscle that has undergone prolonged periods of contraction releases lactate into the circulation.

Heart cells are always well oxygenated. Cardiac cells are energy molecule “scavengers”, they take up the lactate released from muscle cells & red blood cells (see below) and convert it into pyruvate. The H subunit of Lactate dehydrogenase has a low Km, high affinity for lactate and a high Km, low affinity for pyruvate. With this arrangement of Km’s and subunit compositions (H4), heart Lactate dehydrogenase catalyzes the formation of pyruvate from lactate very efficiently. The pyruvate to lactate reaction is very unfavorable in this tissue. Pyruvate in the heart is used as a carbon source for the final common pathways (TCA and ET/OxPhos). During periods of prolonged or rapid skeletal muscle contraction, the waste product generated by the skeletal muscle can be / is used to feed the heart, freeing up glucose for muscle contraction.

Red blood cells (RBC) always function “anaerobically”. These cells do not contain the two final common pathways, the pathways that require oxygen. Red blood cells do not contain mitochondria the site / cellular

TissueMost Abundant Form of LDH

Isotype

Heart H4 LDH1

Kidney H3M & H4 LDH1 & LDH2

Red Blood Cells H3M LDH2

Brain H3M & H2M2 LDH2 & LDH3

White Blood Cells H2M2 LDH3

Skeletal Muscle HM3 & M4 LDH4 & LDH5

Liver M4 LDH5


location of the two final common pathways. The only ATP generating pathway in red blood cells is glycolysis. In red blood cells the pyruvate from glycolysis is converted to lactate to regenerate NAD+ and sustain glycolysis. The lactate that is produced is released from the RBC into the plasma and it is transported primarily to the liver, but the heart takes up some of it. Lactate dehydrogenase in the RBC is predominantly H3M (low Km, high affinity for lactate and a high Km, low affinity for pyruvate) which should favor the conversion of lactate to pyruvate. However, in the RBC the conversion of pyruvate to lactate is favored because the concentrations of pyruvate and lactate are near their equilibrium positions and the constant transport of lactate from the RBC pulls the Lactate dehydrogenase reaction toward the conversion of pyruvate to lactate (Le Chatelier’s Principle).

A similar condition exists in the liver. The liver constantly absorbs the lactate released into the blood by the red blood cell, converts it to pyruvate, and uses the pyruvate for energy generation or glucose synthesis. Liver Lactate dehydrogenase is predominantly the M4 isoenzyme, (low Km, high affinity for pyruvate and a high Km, low affinity for lactate), a form which favors lactate formation. In the liver the concentrations of pyruvate and lactate are near their equilibrium positions. Conversion of lactate to pyruvate occurs in the liver because as the pyruvate is utilized by other pathways the decrease in pyruvate concentration pulls the Lactate dehydrogenase reaction toward pyruvate formation (Le Chatelier’s Principle) even though the M4 isoenzyme favors the reverse reaction, the conversion of pyruvate to lactate.

Control of Glycolysis - Allosteric Regulation

The enzymes that catalyze the three irreversible steps of glycolysis; Hexokinase I, II, & III, Phosphofructokinase, and Pyruvate kinase, are all allosteric enzymes and are all regulatory points of glycolysis.

Hexokinase I, II, & III are allosterically inhibited by glucose-6-phosphate. When the concentration of glucose-6-phosphate is high, it binds to an allosteric site away from the active site and inhibits the activity of the enzyme.

One of the functions of the liver is to rapidly clear glucose from the blood after a carbohydrate rich meal. The primary hexokinase in liver cells is Hexokinase IV (Glucokinase) and this enzyme in conjunction with the GluT2 glucose transporter functions in the liver to accomplish this task. The GluT2 transporter rapidly equilibrates the concentration of glucose in the hepatocyte with the concentration in the blood. When the glucose concentration is low in the blood, it is equally low in liver cells and under these conditions fructose-6-phosphate (F6P) binds to a REGULATORY PROTEIN. The F6P•REGULATORY PROTEIN complex binds to Glucokinase and this ternary complex is transported and sequestered in the nucleus preventing the liver from using glucose when it is scarce. After a carbohydrate rich meal, when glucose concentrations are high the GluT2 transporter rapidly moves glucose into the hepatocyte maintaining equal concentrations across the hepatocyte membrane. Under these conditions, glucose binds to the REGULATORY PROTEIN causing the release of Glucokinase from the REGULATORY PROTEIN and the nucleus. Once activated, Glucokinase phosphorylates glucose at a rate proportional to the glucose concentration in the blood, remember the KM of Glucokinase for glucose is about 10 mM so this enzyme is never saturated under cellular conditions.


Pyruvate kinase is allosterically inhibited by high cellular concentrations of: - ATP - High concentrations of ATP signal an energy rich state within the cell and glycolysis can slow

down. - Acetyl-Coenzyme A - The two carbon acetate (CH3COO–) ion carried by Coenzyme A (acetyl-

Coenzyme A; acetyl-CoA) is the carbon source, the “fuel” for the final common pathways. The acetate fragment is oxidized by these pathways with the concomitant generation of large amounts of ATP.

- Fatty Acids - Fatty acids are better energy source than glucose. When fatty acid concentrations are

Glucose

Glucose-6-phosphate



Phosphoenolpyruvate

Pyruvate

HexokinaseI, II, III

Phosphofructokinase-1

Pyruvate Kinase

Phosphofructokinase-2

ATPADP

kinase activity

H2OPO4

–3

phosphatase activity

Glucokinase (Hexokinase IV)


elevated in the cell it is a signal indicating that blood glucose concentrations are dropping and/or low. The cell will use fatty acids for energy rather than glucose, glycolysis is inhibited, and the little available glucose is saved for the tissues that absolutely require it.

- Alanine - Alanine in one step can be transaminated (the amino group on alanine is transferred to an acceptor molecule) to pyruvate and this pyruvate used in the final common pathways for energy generation. When alanine concentrations are high in the cell, especially skeletal muscle cells, it is a signal indicating that glucose concentrations are dropping and/or low To conserve glucose, glycolysis is inhibited and energy is generated from the available excess alanine.

Pyruvate kinase is allosterically activated by high cellular concentrations of: AMP - High concentrations of AMP signal an energy poor state and glycolysis is stimulated. Fructose-1,6-bisphosphate - Activation by fructose-1,6-bisphosphate is an example of feed forward activation. High concentrations of this intermediate signals pyruvate kinase to pull the reversible reactions in the middle of glycolysis toward completion.

Phosphofructokinase-1 is the primary controlling enzyme of glycolysis. It catalyzes the overall rate limiting step of the pathway. Fructose-1,6-bisphosphate, once formed, is committed to glycolysis This enzyme is allosterically inhibited by high cellular concentrations of:

- ATP - High concentrations of ATP signal an energy rich state within the cell and the rate of glycolysis can be slowed down.

- Citrate - Citrate is the first product of the first final common pathway. When its concentration is high it signals that the final common pathways are running at an elevated rate to generate ATP and glycolysis can be slowed down.

Phosphofructokinase-1 is allosterically activated by high concentrations of: AMP - High AMP concentrations indicate an energy poor state and the energy generating pathways need to be stimulated. Fructose-2,6-bisphosphate - Fructose-2,6-bisphosphate is the primary allosteric effector of Phosphofructokinase-1 (PFK-1) and it has only one function in the cell and that is to stimulate PFK-1. It is synthesized by the action of Phosphofructokinase-2 using fructose-6-phosphate as substrate. It is synthesized when energy supplies are low and hydrolyzed by Phosphofructokinase-2 when energy supplies are high.

Phosphofructokinase-2 is a BIFUNCTIONAL enzyme. It contains two different active sites and catalyzes two different reactions. One active site contains the Kinase activity, this site catalyzes the formation of fructose-2,6-bisphosphate from ATP and fructose-6-phosphate. The other active site is a Phosphatase (Fructose-6-phosphate Phosphohydrolase) activity, this site catalyzes the hydrolytic removal of phosphate from fructose-2,6-bisphosphate to (re)form fructose-6-phosphate and PO4–3.

Allosteric effectors determine which activity of Phosphofructokinase-2 predominates. High cellular concentrations of:

• inorganic phosphate. Concentrations of PO4–3 increase under energy poor conditions. PO4–3 • stimulates the Kinase Activity • inhibits the Phosphatase Activity.

• citrate. Citrate concentration increase during energy rich times. Citrate • stimulates the Phosphatase Activity • inhibits the Kinase Activity.


When the kinase activity is stimulated, the cellular concentrations of fructose-2,6-bisphosphate increases, this increase in fructose-2,6-bisphosphate stimulates Phosphofructokinase-1, and glycolysis is stimulated. When the phosphatase activity is stimulated, the cellular concentration of fructose-2,6-bisphosphate drop, this decrease in fructose-2,6-bisphosphate concentration removes the stimulation of Phosphofructokinase-1, and glycolysis is inhibited (unstimulated).

The ATP:AMP ratio is a manifestation of the energy charge within the cell. As energy is expended the ATP concentration drops and the AMP concentration increases dramatically by the action of adenylate kinase. As the concentrations of AMP increases, some of it binds to and activates a protein kinase - AMP-Activated Protein Kinase or AMPK. (AMP-Activated Protein Kinase, AMPK, is different from cAMP Dependent Protein Kinase, PKA.) Once activated, AMPK catalyzed phosphorylation of key enzymes rapidly switches

ATP ADP

ADP + ADP ATP + AMP

Metabolism

Adenylate

Kinase

Skel

etal

Mus

cle

Car

diac

Mus

cle

Liv

er

GluT4movement &insertion into

plasma membrane- increased glucose

transport

PhosphorylatesPhosphofructokinase-2

cardiac isoenzyme- stimulating kinaseactivity & inhibiting

phosphatase- stimulates glycolysis

PhosphorylatesGlycogen Synthase- inhibits glycogen

synthesis


metabolism from ATP consumption to ATP production. This is a rapid and short-term regulatory process. The effects of AMPK appear to be tissue specific. In skeletal muscle AMPK stimulates the movement of GluT4 transporters from their site of sequestration and their insertion into the plasma membrane. This transport of Glut4 transporters is independent of Insulin. AMPK phosphorylates the cardiac isoenzyme of Phosphofructokinase-2 (PFK2) stimulating the kinase activity and inhibiting the phosphatase activity. Only the cardiac isoenzyme of PFK2 contains an AMPK phosphorylation site, isoenzymes of other tissues do not. This increases the rate of glycolysis and ATP production in heart muscle especially under hypoxic conditions (myocardial infarction). In the liver AMPK phosphorylates and inhibits Glycogen Synthase.

Picture glycolysis “starting up”. The cell is in an energy poor state; the concentration of AMP and PO4–3 is high and the concentration of fructose-6-phosphate is low. Phosphofructokinase-2 (PFK-2) is present in the cell in significantly lower concentration than Phosphofructokinase-1 (PFK-1). When PO4–3 is high PFK-2 is allosterically stimulated and it has a higher affinity for fructose-6-phosphate than unstimulated PFK-1. A large percentage of the fructose-6-phosphate initially formed is converted to fructose-2,6-bisphosphate by the activated PFK-2. As the concentration of fructose-2,6-bisphosphate increases it binds to PFK-1 and stimulates this enzyme. The stimulated form of PFK-1 has a Km for fructose-6-phosphate only slightly smaller (slightly higher affinity) than PFK-2, but since PFK-1 is present in a thousand fold excess or greater over the concentration than PFK-2, once PFK-1 is stimulated the vast majority of fructose-6-phosphate is acted upon by PFK-1 and glycolysis proceeds at a stimulated rate.

Control of Glycolysis - Hormonal Regulation

Hormones also play a role in controlling the rate for glycolysis.

The hormone INSULIN directly and indirectly increases the rate of glycolysis. Insulin directly increases the rate of glycolysis by stimulating the synthesis of Hexokinase, Phosphofructokinase-1, Pyruvate Kinase, and Phosphofructokinase-2. Protein Kinase B phosphorylates transcription factors that stimulate the transcription and translation of the genes for these proteins. Insulin indirectly stimulates glycolysis by two mechanisms. By stimulating the mobilization and insertion of glucose transporters (Glut 4) into the membrane, the cell is able to take up more glucose and the rate of glycolysis is increased, more substrate results in increased reaction rates. Insulin, by stimulating protein synthesis and other anabolic pathways, increases ATP utilization by the cell. The increase in ATP utilization, results in an increase in AMP concentration, and high AMP concentrations stimulate glycolysis.

The hormone GLUCAGON, via the Gs transduction system and Protein Kinase A, inhibits glycolysis in the liver, and only in the liver. One of the substrates of the active Protein Kinase A in the liver is Pyruvate kinase. The liver isoenzyme of Pyruvate kinase is inhibited by phosphorylation by Protein Kinase A. When the phosphates are removed by a protein phosphatase the activity of the enzyme is restored. The effect of glucagon on Pyruvate kinase activity is apparent in liver cells because these cells have receptors for glucagon and they have a Pyruvate kinase isoenzyme that is a substrate for PKA. GLUCAGON does not cause the phosphorylation and inhibition of Pyruvate kinase in other tissues of the body because they either lack the glucagon receptor, they contain a different isoenzyme of pyruvate kinase, or both.

The hepatic isoenzyme of Phosphofructokinase-2 (PFK2) is likewise under the control of GLUCAGON. When the active Protein Kinase A phosphorylates the hepatic isoenzyme of Phosphofructokinase-2 the


Kinase Activity of PFK2 is Inhibited and the Phosphatase Activity is Stimulated. With the phosphatase activity stimulated the level of fructose-2,6-bisphosphate drops, the activity of phosphofructokinase-1 decreases, and the rate of glycolysis slows. Note: The effect of Protein Kinase A on the liver isoenzyme of PFK2 is the exact opposite of the effect of AMPK on the heart isoenzyme of PFK2.

When PFK2 is dephosphorylated by the action of Phosphoprotein Phosphatase 2A the enzyme is under allosteric control. The activity of PFK2 depends upon the concentration of the allosteric effectors.

High Fructose Corn Syrup and Obesity

High fructose corn syrup has been implicated as one of the causes for the increase in obesity. Ignoring the advertisements, high fructose corn syrup is not a natural product. Corn starch is hydrolyzed to glucose and then a large percentage of the glucose is isomerized to fructose. The sweetness of fructose ranges from 1.17 to 1.75 times that of sucrose, so less can be used to give the same level of sweetness. It has been touted as a low glycemic index sweetener since it doesn’t cause an increase in blood glucose {duh! - its fructose} nor an increase in insulin secretion {again - duh! - its fructose}. Sounds good. However, how fructose is metabolized by the liver makes it more lipogenic (fat forming). Fructose in the liver bypasses the first two control points of glycolysis, one of which is the major control point. Bypassing two of the three control points results in abnormally high concentrations of pyruvate and acetyl-CoA, both of which stimulate lipogenesis.


glycolysis - marquette university -

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