6 glycolysis (3)

Carbohydrate Metabolism

Sources

Initial Absorption and Digestion

Oxidation of Glucose: Glycolysis & Citric Acid Cycle

Synthesis of Glucose: Gluconeogenesis

Glycogen Metabolism

Pentose Phosphate Pathway

Organ Integration of Carbohydrate Metabolism

Note: For chemistry & structures of carbohydrates review

Unit 4 in the Self Teaching Program

Dietary Sources of Carbohydrates (Most abundant biomolecules

on earth)

1

Carbohydrate oxidation is the central energy yielding pathway in most cells.

2

Starch: the nutritional

store of glucose in

plants.

A major source of

dietary glucose.

Disaccharides

Monosaccharides

2 Forms of Starch (nutritional reservoir in plants)Branched

Unbranched

1 6

glu glu1 4

23

α-1,4 glycosidic linkages

α-1,4 & α-1,6 glycosidic

linkages

glu fru

glu glu

gal glu

(30) (1)

1 4

gal

glufru

1 4

Similar to glycogen in

animal cells, but less

extensive branching

in starch.

Initial Digestion of Carbohydrates

1) Mouth: limited breakdown of starch and glycogen occurs;

brief period of contact.

Salivary amylase (an α-1,4 glucosidase);

• Specific for internal bonds; does not breakdown α-1,6-glucosides

or α-1,4 bonds located at branch points.

• Oligosaccharides and some maltose are the products.

2) Stomach: no significant digestive enzymes present.

The GI tract is a barrier for large nutrients. Starch and other

oligosaccharides must be broken down to monosaccharides

prior to entry into cells.


3) Small Intestine: Responsible for most of carbohydrate digestion.

a) Lumen: Pancreatic amylase (its substrate specificity is similar to

that of salivary amylase; hydrolyzes internal α-1,4 linkages);

•Secreted by the pancreatic duct into the duodenum.

•Quantitatively more important than the salivary enzyme.

•Products are: maltose (a disaccharide), maltotriose (a trisaccharide),

and the α-limit dextrins.

•The α-limit dextrins contain approx. 8 glucose units with one or more

α-1,6 branch points. They will be further digested to maltose, maltotriose,

and glucose on the luminal surface of the epithelial cells.


b) Brush Border of the Mucosal Epithelium

Final hydrolysis of di- and oligosaccharides to monosaccharides.

Catalyzed by enzymes on the surface of the small intestinal epithelial

cells. Excess capacity for digestion of starch and sucrose.

Only monosaccharides can be absorbed into intestinal epithelial cells.

Undigested Material

Di-, oligo-, and polysaccharides which are not hydrolyzed,

are not absorbed into the intestinal epithelial cells.

Travel to lower portion of small intestine, metabolized by bacteria which

contain many more saccharidases than do human cells.

Products: short chain fatty acids, lactate, hydrogen gas, methane, CO2

These products cause: fluid secretion

increased intestinal motility

cramps (due to distension of gut and/or

irritant effects on the intestinal mucosa)

Absorption of Monosaccharides

Monosaccharides are absorbed from the brush border membrane of

the intestinal epithelial cells and ultimately into the bloodstream via

the actions of specific transport proteins.

Rate of monosaccharide absorption is enhanced via microvilli (increase

surface area) and the existence of specific transport proteins.

Sugar moves from small intestine intestinal epithelial cell bloodstream

Luminal; Brush

border membrane

Microvilli

Contraluminal

membrane

Na-dependent“ActiveTransport”

“FacilitatedDiffusion”Na-independent

“FacilitatedDiffusion”Na-independent

Carbohydrate Maldigestion Disease

Three categories of carbohydrate intolerances:

1) Congenital absence of an enzyme (lactase deficiency) or transport

system: (e.g. glucose-galactose malabsorption syndrome due to a

decrease or lack of Na-coupled glucose, galactose transporter; SGLT-1).

2) Hereditary, delayed onset deficiency: e.g., delayed onset of lactase

deficiency.

Lactose intolerance: frequently seen among adults in which ingestion of

lactose-containing foods (milk products) leads to abdominal cramps and

diarrhea.

Due to disappearance of lactase after childhood; lactose is incompletely

digested and absorbed. Unabsorbed lactose is converted into toxic

products by bacterial enzymes in the large intestine.

3) Acquired Deficiency: as a result of intestinal disease; arises due to

gastroenteritis and destruction of brush border tissue.

Glycolysis (Embden-Meyerhof Pathway)

1) Represents the central pathway for the catabolism of glucose and

other monosaccharides.

2) A nearly universal pathway in biological systems and occurs

in virtually every tissue.

3) The liver is the principal organ in the maintenance of blood glucose

levels. Thus the functional state of the liver will profoundly influence

carbohydrate metabolism.

4) D-glucose is oxidized to pyruvate, which under anaerobic

conditions may then be partially converted to lactate.

Under anaerobic conditions: glycolysis consists of 11 coupled reactions

with the overall net reaction being:

D-glucose + 2 Pi + 2 ADP 2 lactate + 2 H+ + 2 ATP + 2 H2O

ΔG˚’ = -32.4 kcal/mol

Under aerobic conditions: glycolysis consists of 10 coupled reactions

with the overall net reaction being:

D-glucose + 2 Pi + 2 ADP + 2 NAD+

2 pyruvate + 2 H+ + 2 ATP + 2 NADH + 2 H2O

ΔG˚’ = -20.4 kcal/mol

Glycolysis can be divided into 2 stages:

Stage 1 (The Preparatory Phase): Consists of the collection of sugars

by the liver.

Subsequent phosphorylation of these sugars at the expense of 2 ATP’s.

Conversion to glyceraldehyde 3-phosphate.

Stage 2 (The Payoff Phase): Consists of conversion of glyceraldehyde

3-phosphate to either pyruvate or lactate via a series of oxidation-

reduction steps.

Concomitant conservation of energy via formation of 4 ATPs

(5 or 6 enzymes are utilized, respectively).

Stage 1

-2 ATP

Stage 2

+4 ATP

2 NAD+

Lactate (2)

11

2 NAD+

Stage 2

+4 ATP

Stage 1

-2 ATP

Glycolysis: Stage 1

Initial Strategy: Trap glucose in the cell and convert it to a compound

that can be cleaved into phosphorylated 3-carbon units.

1st Reaction: Glucose enters cell and is phosphorylated to glucose

6-phosphate, a negatively charged molecule which is trapped inside

the cell.

6 6

Important Additional Points

1) Reaction is irreversible.

2) Liver also contains a specialized form of hexokinase known as

glucokinase.

3) Glucokinase: much higher Km for glucose (5 mM vs. 0.1 mM)

is not inhibited by glucose 6-phosphate

is absent in muscle and is deficient in patients

with diabetes

4) At normal blood glucose concentrations hexokinase is fully

saturated, glucokinase is not.

5) Glucokinase is present at high concentration in liver and is induced

in response to D-glucose.

6) Glucokinase assures that at high concentrations, glucose is

not wasted. Instead it is converted to glucose 6-phosphate for

subsequent synthesis of glycogen.

7) Hexokinase can also phosphorylate fructose, mannose, and

glucosamine, whereas glucokinase cannot. However, due to its

high Km for fructose, and existence of a separate pathway for

fructose, it is not a significant substrate for hexokinase.

8) First reaction does not commit glucose to glycolysis, since

glucose-6-phosphate represents a branch point in carbohydrate

metabolism. It also enters pentose phosphate pathway and

glycogenesis.

2nd Reaction: the isomerization of glucose 6-phosphate to fructose

6-phosphate.

Catalyzed by phosphoglucose isomerase.

6 membered

pyranose ring

5 membered

furanose ring

1

23

4

5

6

1

2

34

5

6

This reaction is readily reversible.

This reaction represents an example of a conversion of an aldoseto a ketose.

1

2

3

4

5

6

Aldehyde

Ketone

Open chain representation of the sugars.

An aldehyde containing sugar. A ketone containing sugar.

1

2

3

4

5

6

3rd Reaction: Fructose 6-phosphate is phosphorylated by ATP to form

fructose 1,6-bisphosphate.

This is the second of the two priming reactions in glycolysis.

Catalyzed by phosphofructokinase (PFK; PFK1).

PFK is the major point of regulation in glycolysis. Rx is irreversible.

PFK is regulated allosterically.

1

2

34

5

6

4th Reaction: Cleavage of fructose 1,6-bisphosphate to glyceraldehyde

3-phosphate and dihydroxyacetone phosphate.

Represents cleavage of a hexose into two trioses.

Note: Reaction is readily reversible. It is pulled to the right via removal

of glyceraldehyde 3-phosphate via subsequent steps.

Only glyceraldehyde 3-phosphate is on the direct pathway of glycolysis.

1

2

3

4

5

6

1

2

3

4

5

6

5th Reaction: Isomerization of 3-carbon phosphorylated sugars.

catalyzed by triose phosphate isomerase.

1

2

3

4

5

6

Additional Points:

1) At equilibrium 96% of triose phosphate is dihydroxyacetone

phosphate. Rx. is pulled to the right via rapid removal of

the product.

2) Net result: Dihydroxyacetone phosphate is funneled into the main

glycolytic pathway;

2 molecules of glyceraldehyde 3-phosphate are formed from 1

molecule of fructose 1,6-bisphosphate.

3) Triose phosphate isomerase accelerates isomerization by a factor

of 1010. Only limited by diffusion of the substrate into the active site.

Considered a kinetically perfect enzyme.

With this reaction, carbons 1, 2, and 3 of the starting glucose become

indistinguishable from carbons 6, 5, and 4, respectively.

Also, the numbering of carbon atoms in glyceraldehyde 3-phosphate

is not the same as the numbering of carbons in glucose.

Glycolysis: Stage 2

Stage 1: 1 molecule of glucose 2 molecules of glyceraldehyde

3-phosphate.

Stage 2: 2 molecules of glyceraldehyde 3-phosphate

2 molecules of pyruvate

AND

4 ADP 4ATP.

2 NAD+

Stage 1

-2 ATP

2 NAD+

Stage 1

-2 ATP

Stage 2

+4 ATP

Lactate (2)

11

2 NAD+

6th Reaction: Oxidation of glyceraldehyde 3-phosphate to

1,3-bisphosphoglycerate.

The first of the two energy-conserving

reactions of glycolysis that will ultimately

yield ATP.

This is a mixed anhydride

of phosphoric acid and a

carboxylic acid.

Note: The mixed anhydride

has a very high free energy

of hydrolysis.

1

2

3

Aldehyde

group

NAD+-dependent

Mixed

anhydride1

2

3

7th Reaction: First ATP-generating step. ATP is formed as the phosporyl

on the carboxyl group of 1,3-bisphosphoglycerate is transferred to ADP.

“Substrate Level Phosphorylation”

Note: The consequences of this reaction in

combination with the 6th reaction are:

1) An aldehyde is oxidized to a carboxylic

acid group.

2) NAD+ is concomitantly reduced to NADH.

3) ATP is formed from Pi and ADP.

1

2

3

1

2

3

8th Reaction: The phosphoryl group is shifted from the C-3 to the C-2

position of glycerate. Catalyzed by phosphoglycerate mutase.

Note: A mutase transfers a functional group from one position to

another on the same molecule.

1

2

3

1

2

3

9th Reaction: A dehydration reaction is which water is reversibly

removed from 2-phosphoglycerate to from phosphoenolpyruvate.

Catalyzed by enolase.

Large difference in the standard free energy of hydrolysis of the

phosphate group in the reactant versus the product.

1

2

3

1

2

3

10th Reaction: Transfer of a phosphoryl group from PEP to ADP

catalyzed by pyruvate kinase.

Irreversible; An important site of regulation in the liver.

The second “substrate level phosphorylation”.

11th Reaction: Reduction of pyruvate to lactate via the enzyme

lactate dehydrogenase.

Conversion of occurs under partially anerobic conditions, when

oxygen is limited (e.g., muscle during intense activity) OR in certain

tissues even when sufficient oxygen is present (retina, brain, RBCs).

NADH required for this reaction is supplied

by the 6th reaction (the dehydrogenation of

glyceraldehyde 3-phosphate).

Importantly, under anaerobic conditions, theregeneration of NAD+ by this step is essentialfor the continued functioning of glycolysis.

Stage 1

-2 ATP

Stage 2

+4 ATP

Lactate (2)

11

2 NAD+

There are 5 isozymic forms of lactate dehydrogenase.

Differ in their affinity for substrate and sensitivity to allosteric

inhibition.

Isozymes: multiple forms of a given enzyme that catalyze the

same reaction but differ in kinetic or regulatory properties.

Each LDH isozyme contains 4 copes of two different polypeptides.

H form (heart) and M form (muscle). Designated H4, H3M1, H2M2, etc.

H4: higher affinity for substrate; allosterically regulated.

Designed to oxidize lactate to pyruvate which can be used by

the heart as an aerobic fuel source.

M4: optimized to convert pyruvate to lactate in muscle; allows

glycolysis to continue under anaerobic conditions.

Net reaction in transformation of glucose into pyruvate:

D-glucose + 2 Pi + 2 ADP + 2 NAD+

2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

2 ATPs are generated during conversion of glucose to 2 pyruvate.

3 Reactions are irreversible under physiological conditions:

hexokinasephosphofructokinasepyruvate kinase

Entry of Other Monosaccharides into Glycolysis

D-Fructose: present in fruits; also can be generated by hydrolysis of

sucrose (yield fructose + glucose).

Note: In the liver hexokinase has a 20x higher affinity for glucose

compared to fructose. Since there is a lot of glucose present in this

organ, fructose is not principally metabolized by hexokinase, but

rather by the following pathway:

(1)(2)

(1) Fructose intolerance results from a deficiency in fructose 1-phosphate

aldolase. Leads to an accumulation in fructose 1-phosphate and a depletion

of ATP and Pi. Pi depletion makes it impossible to generate more ATP

lowering levels even further. Causes cell damage.

(2) Fructosuria results from a deficiency in fructokinase. Fructose appears

in blood and urine. Relatively benign metabolic abnormality.

D-mannose: arises from digestion of polysaccharides and glycoproteins

found in food.

Mannose + ATP Mannose 6-phosphate

Mannose 6-phosphate Fructose 6-phosphate

Hexokinase

Phosphomannose isomerase

D-Galactose: Derived via hydrolysis of

the disaccharide lactose.

It is converted to glucose 1-phosphate

as follows:

Step 1: galactose is phosphorylated.

Step 2: Galactose 1-phosphate acquires

a uridyl group from UDP-glucose with a

release of glucose 1-phosphate.

Step 3: The galactose moiety of UDP-galactose

is epimerized to glucose; the configuration

of the hydroxyl group at C-4 is inverted.

Net effect is conversion of galactose 1-phosphate to glucose 1-phosphate.

1

UDP

Glucose 1-phosphate is then isomerized to glucose 6-phosphate:

Glucose 1-phosphate Glucose 6-phosphatephosphoglucomutase

•The disease galactosemia results from the absence of the enzyme

galactose 1-phosphate uridyl transferase.

•Galactose metabolism is blocked at the galactose 1-phosphate step.

•Damage occurs due to the accumulation of toxic substances rather

than due to the absence of an essential compound.

One of the offending compounds is galactitol

which is produced by the reduction of

galactose.

•Galactosemia is a severe disease.

•Symptoms occur when milk is consumed; liver

becomes enlarged; jaundice is common.

•Blood galactose is elevated and it’s found

in the urine is well.

•Absence of the transferase enzyme from RBCs

is diagnostic.

•Treatment involves exclusion of galactose from

the diet.

Conversion of Glucose to Fructose via Sorbitol

•Aldose reductase reduces glucose to sorbitol, which is quite polar

and thus does not passively diffuse across membrane.

•Also, its not a substrate for the glucose transporter.

•Therefore its trapped inside cells.

•Liver, ovaries, sperm, and seminal

vesicles contain the enzyme sorbitol dehydrogenase.

•Oxidizes sorbitol to fructose.

•Fructose then enters glycolysis or

gluconeogenesis.

•When glucose is elevated (e.g., diabetes) and if there is sufficient

NADPH, aldose reductase produces excess sorbitol.

•Retina, lens, kidney, and nerve cells do not contain sorbitol dehydrogenase and therefore sorbitol accumulates.

•Causes a strong osmotic effect and cell swelling due to water retention.

•Symptomalogy occurs (cataract formation, peripheral neuropathy, and

vascular problems).

No sorbitol

dehydrogenase.

Dietary Disaccharides are Hydrolyzed to Monosaccharides

Disaccharides cannot directly enter cells without first being hydrolyzed

to monosaccharides (extracellularly). Hydrolysis reactions are

catalyzed by enzymes attached to outer surface of epithelial cells

lining the small intestine.

The resulting monosaccharides enter cells lining the intestine via

specific transport proteins. Then pass from cells into the blood,

distributed to the liver, enter the glycolytic pathway.

Regulation of Glycolysis

Enzymes catalyzing irreversible reactions are often potential control

sites.

In glycolysis regulation occurs at hexokinase, phosphofructokinase,and pyruvate kinase.

I. Phosphofructokinase: the most important control point in

glycolysis. It is the first irreversible reaction that is unique to the

pathway (i.e., the committed step).

• Inhibited by ATP

• AMP reverses the inhibition by ATP. Thus PFK activity increases

when the ATP/AMP ratio is lowered (i.e., as the energy charge of

the cell decreases).

• Inhibited by a decrease in pH.

• Inhibited by citrate.

• Activated by Fructose 2,6-bisphosphate.***

•Fructose 2,6-BP is formed by phosphorylation of fructose 6-phosphate

via the enzyme phosphofructokinase 2.

•Fructose 2,6-BP can be hydrolyzed by the enzyme fructose bisphosphatase 2.

•Fructose 6-phosphate both accelerates the synthesis of fructose 2,6-BP

and inhibits its hydrolysis. Both mechanisms lead to increased fructose 2,6-BP.

Phosphofructokinase 2

Fructose bisphosphatase 2

Causes phosphorylation of

the enzyme which then

activates the phosphatase

function.

16

1

•The activities of PFK2 and FBPase2 reside on the same polypeptide

chain.

•Both activities are reciprocally regulated by phosphorylation of a

single serine residue.

Thus low blood glucose, blood glucagon, cAMP-dependent

phosphorylation of this bifunctional enzyme, PFK2 and FBPase 2,

which then F 2,6-BP, and the activity of PFK1.

Phosphofructokinase 2

Fructose bisphosphatase 2

Causes phosphorylation of

the enzyme which then

activates the phosphatase

function and inhibits the

kinase.

Net result is to decrease fructose 2,6-bisphosphate

level and therefore decrease PFK1 activity.

II. Hexokinase: inhibited by its product glucose 6-phosphate. Thus

PFK inhibition leads to hexokinase inhibition via the buildup of

metabolites.

III. Pyruvate Kinase: catalyzes the third irreversible step in glycolysis.

Controls the outflow of the pathway.

2 forms: L form (liver): subject to extensive allosteric regulation.

Fructose 1,6-bisphosphate activates.

ATP and alanine inhibit.

M form (muscle and brain): not allosterically regulated.



The L form of pyruvate kinase is inhibited by hormone-mediated

cAMP-dependent phosphorylation as depicted below:

Low glucose stimulates phos-

phorylation of pyruvate kinase

which inactivates the enzyme.

Low glucose inhibits

dephosphorylation of

pyruvate kinase thereby

maintaining the inactive

form of the enzyme.

Low blood glucose, glucagon, cAMP-dependent phosphorylation,

of pyruvate kinase, INACTIVATES.

Thus low blood glucose, PFK1 and pyruvate kinase.

Bottom Line: liver does not consume glucose when it is more urgently needed by brain and muscle.

Alcoholic Fermentation

The sequence of reactions from glucose to pyruvate is similar in all

organisms.

However, in yeast and several other microorganisms ethanol is

formed from pyruvate via the following 2 reactions:

Note: the CO2 produced via pyruvate

decarboxylation in Brewer’s yeast is

responsible for the carbonation of

champagne.

In baking the CO2 released when yeast

is mixed with a fermentable sugar

causes the dough to rise.

Absent in

Humans.

Present in humans.

partly responsible

for the oxidation of

ethanol.

Education

6 glycolysis (3)