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Carbohydrates Carbohydrates are hydrates of carbon (CH2O)n, n>3

Sugars:
Monosachharides simple sugars (can not be broken down to simpler sugars under mild conditions)

Oligosachharides a few (2 to 10) monosachharides linked by glycosidic bonds


Polysachharides polymers of 20 or more monosachharides

Monosachharides .

2 :
- aldose ( , ) - ketose ( , )

Polysaccharides

Starch and glycogen are storage molecules

Chitin and cellulose are structural molecules

Cell surface polysaccharides are recognition molecules

Storage Glycogen The glucose storage device in animals (present in all cells)

Glycogen constitutes up to 10% of liver mass and 1-2% of muscle mass Glycogen is stored energy for the organism Only difference from starch: number of branches Alpha(1,6) branches every 8-12 residues Like amylopectin, glycogen gives a red-violet color with iodine

Glycogen deposits in liver

Starch

Storage

Composed of amylose (30%) and amylopectin (70%)


Chloroplast

Amylose is a linear polymer of glucose and poor soluble in water. Forms helical structure Is a storage molecule in plants Why good for storage? Glucose monomers diffuse in the cell. Too much glucose would cause osmotic pressure. Therefore, cells arrange the glucose in long polymers. When glucose is required the enzymes remove monomers from the ends.

Starch stored in amyloplasts

Amylopectin branched polymer of glucose which provide a mechanism for quickly releasing (or storing) of glucose units for (or from) metabolism

Starch

In amylose - iodine fits into the helices to produce a blue color, a way of staining cells when testing for starch.

Amylopectin - Does not form helices because branches every 24-30 residues and need 6 residues per turn in a helix.

Storage Glycogen

Structure: Similar to amylopectin but more branches, that allow rapid mobilization of glucose when required.

Glycogen

Glycogen is a polymer of glucose residues linked by a(14) glycosidic bonds, mainly a(16) glycosidic bonds, at branch points. Glycogen chains & branches are longer than shown.
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Polysaccharides

Structure

Plants rely on polysaccharides for structure. Do not synthesize keratin and collagen.
Cellulose Cellulose consisting of a linear chain of several hundred to over ten thousand linked glucose units Half the carbon in the biosphere is in the form of cellulose Cellulose is the most abundant natural polymer on earth Provides strength for plants and trees But can also be soft in cotton Plant cell wall

Cellulose

Why is it a structural molecule?

Structure

The 1-4 link causes a fully extended linear polymers and can form hydrogen bond with polymers that are parallel to it Animal enzymes do not have enzymes that cleave the 1-4 bond. Therefore humans can not utilize the vast cellulose around them. Cow intestines have symbiotic bacteria that can digest cellulose. Slow process.
Converting cellulose from energy crops into biofules is under investigation as an alternative fuel source .

Structure

Cellulose

Form sheets

Catalytic site
Binding site

Substrate

Enzyme

+ Enzyme

ES Substrate Complex


The product is finally made and the enzyme is ready for another substrate. EP E + P

- Ea


- , , . , . . , .
- . , , . .

( ) . ' .ATP %04 ATP

ADP + P + Energy ---> ATP ATP --> ADP + P + Energy


ATP .
ATP

ATP

Cells need energy in the form of ATP. How does the cell generate ATP?

ADP + P + Energy ---> ATP

ATP --> ADP + P + Energy


ATP synthesis: - occurs in the cytosol by glycolysis - occurs in mitochondria by cellular respiration - occurs in chloroplasts by photosynthesis

Metabolism The process by which living organisms acquire and use free energy to carry out their functions: Catabolism (degradation) Breakdown of nutrients to get components and/or energy Anabolism (biosynthesis) Biomolecules are synthesized from simpler components

Deoxyribonucleic Acid (DNA)


Made up of nucleotides (DNA molecule) in a DNA double helix.
Nucleotide: Phosphate group 1. 5-carbon sugar 2. Nitrogenous base 3. ~2 nm wide

DNA Nucleotide
Phosphate Group

O O=P-O O

CH2
O

N
C
Sugar (deoxyribose)

Nitrogenous base (A, G, C, or T)

DNA Double Helix


5

P
5 4 3 2 1

O O
1

3
4

P
5

T
O

A
O

DNA Double Helix

BASE-PAIRINGS
H-bonds

PCR

)Glycolysis = sweet splitting (Greek 3 - pyruvate

ATP- .ADP
. ( ). , .


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Glycolysis

Takes place in the cytoplasm

O CH3 C

Pyruvate COOFermentation in anaerobic organisms

Ethanol

CO2

Energy investment steps


Reaction 1

Glucose enters the cell through a specific carrier transports glucose into the cytosol

The glycolytic enzymes are located in the cytosol. They are loosely associated with cell structures and membranes.

Glycolysis has 2 phases: Converts glucose to two G-3-P with energy input

Converts glucose to two C3 units (pyruvate) of lower free energy.

The free energy released will be used to make ATP.

Produces two pyruvates, ATP and NADH

Glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP + 2 H2O + 4 H+

Energy investment steps


Reaction 1 The enzyme: Hexokinase

* The kinase activity of the enzyme: transfers a phosphoryl group between ATP and a metabolite

* The reaction is highly spontaneous


* Hexokinase can phosphorylate other sugars: mannose, fructose a non-specific enzyme * Enzyme has low Km, and because glucose levels are usually high it functions in Vmax. * In liver, glucokinase has high Km value so it is more sensitive to the intercellular glucose level

Energy investment steps


Reaction 1 The enzyme: Hexokinase

(Model for induced fit)

Glc

* Glu-6-P can regulate hexokinase activity by inhibition: it can compete for the active site and also by allosteric interactions at another site of HK.

Energy investment steps


Reaction 2 The enzyme: Phosphoglucose isomerase

* Isomerization of an aldose to a ketose

* Because glucose and fructose are cyclic - the isomerization requires ring opening. * The intermediate is called an enediol
* This reaction is reversible

Energy investment steps


Reaction 2 The enzyme: Phosphoglucose isomerase

* Isomerization of an aldose to a ketose * Why is this step needed? Carbonyl oxygen (C=O) is transferred from C1 to C2, and the OH on C1 is accessible for the next phosphorylation. (Would be hard to do on glucose). This also prepares the molecule for cleavage in step 4.

Energy investment steps


Reaction 3 The enzyme: Phosphofructokinase

* 2nd ATP investment. Phosphorylated on two carbons. * Spontaneous and irreversible reaction. * Reaction is similar to HK. * PFK is the rate-limiting enzyme in glycolysis. * Enzyme is highly regulated will be discussed.

Energy investment steps


Reaction 4 The enzyme: Aldolase

Splitting of the sugar = glycolysis

Carbons are renumbered


2 1 2 3 1

4 5 6 1 2

Preparatory stage:

Energy investment steps


Reaction 5 The enzyme: Triose phosphate isomerase (TIM)

* Since only GAP continues in glycolysis, this step is required so all glucose carbons are utilized.

* Since GAP is low in cells (since GAP is used up in the downstream reactions) the reaction is actually exerogenic
Keq = [GAP]/[DHAP] = 4.73 x 10-2 [DHAP]>>[GAP] but because GAP is utilized downstream, more DHAP is converted to GAP

Energy generation steps


Metabolic energy produces 4 ATPs

Net yield in glycolysis: 2 ATPs

Second phase involves two very high energy phosphate intermediates:

- 1,3 BPG (1,3-bisphosphoglycerate )


- Phosphoenolpyruvate

Energy generation steps


Reaction 6 The enzyme: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

* First high energy intermediate is formed acyl phosphate. * The phosphate group has high free energy of hydrolysis: G = -49.4 kJ/mol

* NAD is a co-enzyme that accepts electrons and a H+ from the substrate, and is reduced to NADH.

Energy generation steps


Reaction 6 The enzyme: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Inhibited by iodoacetate and heavy metals

The reaction uses a cysteine thiol at the active site

Energy generation steps


Reaction 6 The enzyme: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Nicotinamide Adenine Dinucleotide (NAD) is a coenzyme derived from the vitamin niacin, or B3. Is important in reduction and oxidation reactions. (oxidation = loose electrons; reduction = gains electrons)

NAD is an electroncarrying molecule.

NAD+ picks up H2 and briefly becomes NADH2+, then quickly stabilizes by losing H+ and becoming NADH

NAD+ accepts 2 e- plus one H+ (a hydride) in going to its reduced form.

Energy generation steps


Reaction 7 The enzyme: Phosphoglycerate kinase

(1,3-BPG)

* First generation of ATP * At this point the net ATP yield is zero (from 2 GAPs)

* (Called a kinase because transfers a phosphoryl between a metabolite and ATP, although it is the reverse reaction)
Reactions 6 (G 0=+6.7) and reaction 7 (G0= -18.8) are coupled G0 =-12.1 kJ/mol

Energy generation steps


Reaction 8 The enzyme: Phosphoglycerate mutase

* Phosphate is shifted from C3 to C2 * Requires magnesium * [3-PG] > [2-PG] so the reaction is actually exergonic in cells

Energy generation steps


Reaction 10 The enzyme: Pyruvate kinase

tetramer

* Spontaneous reaction

* Requires K+ and Mg++


Regulatory site

Active site + ions

Energy generation steps


Reaction 10 The enzyme: Pyruvate kinase

* Removal of Pi from PEP yields an unstable enol, which spontaneously converts to the keto form of pyruvate * Highly exergonic and drives ATP synthesis * Hydrolysis of the phosphate in Phosphoglycerate will give only a G of 17.6 kJ/mol not enough to drive ATP synthesis But PEP has a larger G of phosphate hydrolysis than ATP. (PEP -61 kJ/mol, ATP -31 kJ/mol)

Glycolysis
Balance sheet for ~P bonds of ATP: 2 ATP expended 4 ATP produced (2 from each of two 3C fragments from glucose) Net production of 2 ~P bonds of ATP per glucose.

Glycolysis - total pathway, omitting H+:


glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP

The fate of pyruvate and NADH

The fate of pyruvate and NADH Aerobic or anaerobic: 1) NADH is energy - two possible fates:

* If O2 is available: NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation (1NADH=3ATP)
* In anaerobic conditions: NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

2) Pyruvate is energy - two possible fates: * Aerobic: citric acid cycle * Anaerobic: Animals, bacteria - LDH makes lactate Yeast alcoholic fermentation

The fate of pyruvate and NADH

NADH: must be changed back to NAD+ for glycolysis to continue (little NAD+ in cells) * Aerobic conditions mitochondrial electron transport NADH oxidized & ATP energy
* Anaerobic conditions: 1. Bacteria dont have mitochondria (no respiratory chain) 2. High rates of glycolysis in animal cells, like skeletal muscle during exercise - there is no more oxygen and high demand for ATP, and not enough energy to support it Pyruvate is converted to lactate, regenerating NAD+ needed for glycolysis, the main source of ATP under anaerobic conditions.

3. Alcoholic fermentation

The fate of pyruvate and NADH

Lactic acid fermentation

Pyruvate is reduced to lactate & NAD+

* Lactate diffuses from tissue to blood and transported to heart and liver, that convert it back to glucose * High lactate in muscle does not cause fatigue, but instead the buildup of acid. As a result pH drops and causes Bohr effect in order to increase oxygen delivery to the tissues.

The fate of pyruvate and NADH Lactic acid fermentation

Lactate dehydrogenase
Tetrameric enzyme, composed of two subunits M and H. Enzyme exists in different tissues as isozyme (different aa sequence of the enzymes that catalyze the same reaction). Two subunits can randomly bind one another, so it can be 5 isozymes: M4, M3H, M2H2, M2H3, H4.Each isozyme is specific for given tissue. Clinical significance: LDH pattern in serum can tell us about pathological condition

Control of glycolysis
Control points: Enzymes that catalyze reactions that are highly exerogenic (irreversible) Allosteric enzymes Large negative free energy changes are found in only: 1) Hexokinase 2) Phosphofructokinase 3) Pyruvate kinase

HK

PFK

PK

( ) = micromolar concentrations

Control of these enzymes determines the rate of the glycolysis pathway

Control of glycolysis

* HK activity is controlled by its product: glucose-6-phosphate

* Phosphorylation of glucose ensures that it will stay in the cells.


* If there is enough glucose in the cell: Accumulation of the G6P product ensures that cells will not continue to import glucose.

Control of glycolysis
In liver cells there is a hexokinase variant: Glucokinase Has a high Km for glucose active only at high glucose.

This allows liver cells to store glucose as glycogen when blood levels are high. So G6P does not inhibit glucokinase activity (G6P is a substrate for glycogen synthesis).

Control of glycolysis
Glucokinase

When glucose is required in the blood enzyme glucose-6-phosphatase will release glucose to the bloodstream.
Glucokinase

The enzymes Glucokinase & Glucose-6-phosphatase, are both found in liver but not in most other body cells. This allows the liver to control blood glucose levels.

Control of glycolysis
Phosphofructokinase The rate-limiting enzyme in glycolysis

Because: 1) HK has a variant that acts differently in liver 2) Pyruvate kinase is the last step too late for regulation

ATP is both a substrate and an allosteric inhibitor: there are 2 binding sites for ATP in each subunit a substrate site, an inhibitory site.
Citrate is a PFK inhibitor. Activators of PFK: ADP, AMP, fructose-2,6-bisphosphate

Control of glycolysis

Phosphofructokinase

PFK is a tetramer

Each subunit has 2 ATP-binding sites: Substrate site or active site and inhibitory site
At low concentration, ATP binds only at the active site. At high ATP: ATP binds to the inhibitory site (low-affinity regulatory site) and decreases the affinity for its substrate Fructose-6-P

Control of glycolysis

Phosphofructokinase

PFK increases activity when energy status is low PFK decreases activity when energy status is high

At high levels of ATP in a cell the cell does not need any more ATP. Therefore it is logical that the ATP itself inhibits its own generation, and it is more useful for the cell to store glucose as glycogen when ATP is plentiful. The tense conformation of PFK: at high [ATP], has lower affinity for the other substrate, fructose-6-P, and binds ATP at the inhibitory site. Sigmoidal dependence of reaction rate on [fructose-6-P] is seen.

AMP, present at significant levels only when there is extensive ATP hydrolysis, antagonizes effects of high ATP.

Control of glycolysis

Phosphofructokinase

F-2,6-BP is a potent activator of PFK (PFK1). It is not a glycolytic metabolite.

Synthesized in cells by: phosphofructokinase 2 (PFK2)

Even when there is high ATP it allows activity of PFK1

Fructose-6-P
PFK-2 Fructose-2,6-BP cAMP dependent protein kinase ATP - cAMP glucagon FBPase-2

PFK-2/FBPase-2 PFK-2 FPBase-2 PFK-2 FPBase-2

Fructose-2,6-BP

PFK-1

Phosphoprotein phosphatase

Fructose-2,6-BP

PFK-1

Control of glycolysis

Pyruvate kinase

Inhibited by ATP reduces affinity to phosphoenolpyruvate

Inhibited by acetyl-CoA: If there is enough fatty acid breakdown, then dont need glycolysis
Activated by AMP

Activated by Fructose-1,6,-Bisphosphate (product of PFK1): This ensures that if this product is made that it will pass through the full pathway and that intermediates will not accumulate
Dietary regulation of the enzyme level (after reach carbohydrate meal enzyme level increases up to 10 folds)
cAMP-dependent protein kinase phosphorylates pyruvate kinase and inhibits it.

Thus glucagon-induced production of cyclic-AMP results in inhibition of glycolysis in liver, by slowing of both pyruvate kinase and phosphofructokinase

Feeder pathways for glycolysis


Many carbohydrates besides glucose meet their catabolic fate in glycolysis, after being transformed into one of the glycolytic intermediates.

Feeder pathways for glycolysis

Glycogen and starch, polymeric storage forms of glucose, enter glycolysis in a two-step process: 1) Phosphorolytic cleavage of a glucose residue from an end of the polymer, forming glucose 1phosphate, is catalyzed by glycogen phosphorylase or starch phosphorylase. 2) Phosphoglucomutase then converts the glucose 1-phosphate to glucose 6-phosphate, which can enter glycolysis.

Feeder pathways for glycolysis Ingested polysaccharides and disaccharides: Disaccharides must be hydrolyzed to monosaccharides before entering cells. Intestinal disaccharides and dextrins are hydrolyzed by enzymes attached to the outer surface of the intestinal epithelial cells:

Feeder pathways for glycolysis D-Galactose is formed mainly by hydrolysis of lactate (milk). D-galactose is converted in a number of reactions to glucose-6-phosphate

Galactosemia a genetic disorders in galactose usage. As a result galactose is accumulated and causes eye cataracts, decrease in mental development and other problems. The disease is found mainly in infants.
Lactase is an enzyme that catalyses lactose breakdown to glucose and galactose. In some individuals enzyme disappears from the small intestine cells at the age 4-6. This condition is called lactose intolerance

Animations http://programs.northlandcollege.edu/biology/Biology1111/animations/glycolysis.html

http://www.iubmb-nicholson.org/swf/glycolysis.swf

http://www.maxanim.com/biochemistry/How%20Glycolysis%20Work/How%20Glycoly sis%20Work.htm

How do we get acetyl-CoA?

Acetyl groups for oxidation enter as acetyl-CoA

How do we get acetyl-CoA?


From the carbohydrate pathway under aerobic conditions: The enzyme pyruvate dehydrogenase turns pyruvate into acetyl-CoA through oxidative de-carboxylation

How do we get acetyl-CoA?

Oxidation of pyruvate to acetyl-coA is a multiple step process which involves: - pyruvate decarboxilation - NAD+ reduction - pyruvate oxidation - pyruvate activation by coenzyme A

The reaction is highly exergonic

Pyruvate dehydrogenase
Pyruvate dehydrogenase complex is composed of three enzymes: Pyruvate dehydrogenase (E1) Dihydrolipoamide transacetylase (E2) Dihydrolipoamide dehydrogenase (E3) and five coenzymes Thiamine Pyrophosphate (TPP) Lipoic Acid-Lipoamide FAD NAD+ CoASH

Pyruvate dehydrogenase
In eukaryotes the mitochondrial PDH has: 20 E2 trimers 30 E1 heterotetramers 12 E3 dimers

This is an example in which intermediate products are passed from one enzyme to another. If the enzymes depend only on diffusion then this is very efficient. Also, they are passed to each other and side reactions are minimized. The enzymes can be controlled together.

Five coenzymes are used - TPP, CoASH, Lipoic acid, NAD+, FAD There are 5 reactions, the net stoichiometry is: Pyruvate + CoA + NAD+ acetyl-CoA + CO2 + NADH

Coenzymes - " " Casimir Funk )B1( thiamine


five coenzymes Thiamine )Pyrophosphate (TPP Lipoic Acid-Lipoamide FAD +NAD CoA-SH

1.Thiamine pyrophosphate - TPP

Thiazole ring

Pyrimidine ring

- TPP -keto

Pyruvate dehydrogenase

4. Flavin coenzymes FAD B2 .riboflavin


Isoalloxazine ring system

2 FAD ) ( FADH2 - - ( FAD )riboflavin flavoproteins

Pyruvate dehydrogenase

Pyruvate dehydrogenase multienzyme complex (from Bacillus) rendered from 3D data with lipoyl domains added by hand. The lipoyl arms move the intermediates between the enzymes.

Pyruvate dehydrogenase

Pyruvate dehydrogenase multienzyme complex (from Bacillus) rendered from 3D data with lipoyl domains added by hand. The lipoyl arms move the intermediates between the enzymes.

Pyruvate dehydrogenase

glucose-6-P Glycolysis pyruvate fatty acids acetyl CoA oxaloacetate Krebs Cycle ketone bodies cholesterol citrate

Acetyl CoA functions as: input to Krebs Cycle, where the acetate moiety is further degraded to CO2. donor of acetate for synthesis of fatty acids, ketone bodies, & cholesterol. Since they are no other pathways for making acetyl-coA from pyruvate, this pathway must be regulated

Pyruvate dehydrogenase Regulation of Pyruvate Dehydrogenase Complex by product inhibition: 1) Product inhibition by NADH & acetyl CoA: NADH competes with NAD+ for binding to E3. Acetyl CoA competes with CoA for binding to E2. NADH and acetyl-CoA are products of fatty acid oxidation, so when lipids are available the cell will preserve stores of carbohydrates.

Pyruvate dehydrogenase

Regulation of Pyruvate Dehydrogenase Complex by phosphorylation/dephosphorylation (only in eukaryotes): 2) Phosphorylation of E1 inhibits the complex: A family of Pyruvate Dehydrogenase Kinases catalyze phosphorylation of E1, inhibiting the complex. These kinases are activated by NADH & acetyl CoA (another way the major products inhibit the complex). Amounts of these Kinases increase in muscle mitochondria during starvation, effectively blocking catabolism of glucose (glycolysis), since glucose is needed by the brain.

Pyruvate dehydrogenase Regulation of Pyruvate Dehydrogenase Complex by phosphorylation/dephosphorylation:

A Ca++-sensitive isoform of the phosphatase that removes Pi from E1 , is expressed in muscle cells. Increased cytosolic Ca++ can lead to Ca++ uptake by mitochondria, & activation of Pyruvate Dehydrogenase. Since muscle contraction involves Ca, metabolism may be stimulated during exercise. Pyruvate Dehydrogenase Kinase and Phosphatase enzymes are unique. They bind specifically to Pyruvate Dehydrogenase within the mitochondrial matrix.

Aerobic fate of pyruvate

Citric Acid cycle


(Named after the first product of the cycle - citrate)

Krebs cycle
(Hans Krebs, Nobel prize 1953)

Tri-carboxylic acid (TCA) cycle


(Oxidizes acetyl groups from several sources, not only pyruvate)

Three stages of respiration


1) The citric acid cycle is not just a continuation of glycolysis but is a central pathway for recovering energy from several metabolic fuels: Carbohydrates Fatty acids Amino acids that are broken down to acetylCoA for oxidation.
2) Oxidation of carbons in acetyl groups to produce: CO2, reduced electron carriers (NADH), some ATP 3) Oxidation of electron carriers and more ATP

Citric acid cycle

Cycle:
a multistep cycle in which the first step is regenerated in the last step the cycle can oxidize an unlimited number of acetyl groups

Citric acid cycle

In eukaryotes all the enzyme of the cycle are in the mitochondria. This requires either production of metabolites in the mitochondria or transport out.

Citric acid cycle

Localization of Krebs Cycle: Glycolysis occurs in the cytosol of cells. Pyruvate enters the mitochondrion to be metabolized further.

Mitochondrial Compartments:

The matrix contains Pyruvate Dehydrogenase, enzymes of Krebs Cycle, and other pathways, e.g., fatty acid oxidation & amino acid metabolism. The outer membrane contains large VDAC channels. The outer membrane is leaky to ions & small molecules.

Citric acid cycle

Mitochondrial compartments

Inner membrane infoldings, called cristae, contain constituents of the respiratory chain & ATP Synthase. The inner membrane is the major permeability barrier. It contains various transport catalysts, including a carrier protein that allows pyruvate to enter the matrix.

Pentose Phosphate Pathway

Other names: Phosphogluconate Pathway Hexose Monophosphate Shunt The linear part of the pathway carries out oxidation and decarboxylation of the 6-C sugar glucose-6-P, producing the 5-C sugar ribulose-5-P. Takes place in the cytosol

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Pentose Phosphate Pathway

The energy currency in cells is ATP. But cells also use: reducing power. The Pentose Phosphate Pathway provides:

1) NADPH for reductive biosynthesis of fatty acids or cholesterol (plus ATP)


2) Ribose-5-phosphate for nucleotide and nucleic acid synthesis

The Pentose Phosphate Pathway is an alternative pathway to glycolysis


Involves two steps of oxidation and the rest are non-oxidative

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Pentose Phosphate Pathway


Rapidly dividing cells, such as those of bone marrow, skin, and intestinal mucosa, use the pentoses to make RNA, DNA In other tissues, the essential product of the pentose phosphate pathway is not the pentoses but the electron donor NADPH, needed for reductive biosynthesis or to counter the damaging effects of oxygen radicals. Tissues that carry out extensive fatty acid synthesis (liver, adipose, lactating mammary gland) or very active synthesis of cholesterol and steroid hormones (liver, adrenal gland, gonads) require the NADPH provided by the pathway.

Erythrocytes and the cells of the lens and cornea are directly exposed to oxygen and thus to the damaging free radicals generated by oxygen.
By maintaining a reducing atmosphere (a high ratio of NADPH to NADP and a high ratio of reduced to oxidized glutathione), they can prevent or undo oxidative damage to proteins, lipids, and other sensitive molecules.
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Pentose Phosphate Pathway NADPH and NADH are different molecules: Oxidizing enzymes use NADH (catabolic) Reducing enzymes use NADPH (anabolic)

Occurs mainly in tissues that synthesize fatty acids and steroids: cytoplasm of liver, adipose tissue

Reduction of NADP+ (as with NAD+) involves transfer of 2 e- and 1 H+ to the nicotinamide moiety. This difference between NAD and NADP has little to do with redox activity, but is recognized by substrate-binding sites of enzymes. It is a mechanism for separation of catabolic and synthetic pathways.

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Pentose Phosphate Pathway

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Pentose Phosphate Pathway

Oxidative phase

Obtain: 2 NADPH Oxidation of 1 carbon to CO2 Pentose-Phosphate

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Pentose Phosphate Pathway

G-6-P from: glycolysis or glycogen breakdown


This step is the regulated step The dehydrogenase is inhibited by NADPH. If the availability of NADPH drops due to use in reductive synthetic pathways the PPP is activated.

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Pentose Phosphate Pathway

This reaction occurs without the enzyme. The enzyme increases the rate of hydrolysis.

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Pentose Phosphate Pathway

Obtain: 2 NADPH Oxidation of 1 carbon to CO2 Pentose-Phosphate

The rest of the pathway is non-oxidative

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Pentose Phosphate Pathway

Regulation of Glucose-6-phosphate Dehydrogenase: Glucose-6-phosphate Dehydrogenase is the committed step of the Pentose Phosphate Pathway. This enzyme is regulated by availability of the substrate NADP+. As NADPH is utilized in reductive synthetic pathways, the increasing concentration of NADP+ stimulates the Pentose Phosphate Pathway, to replenish NADPH. NADPH strongly inhibits the enzyme.

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Pentose Phosphate Pathway Two options for Ribulose-5-P: 1) Ribulose-5-P isomerase

Ribose-5-P is the precursor of nucleotides. If the cell makes too much Ribulose-5-P then it is converted to glycolysis (converted to F-6-P and GAP, that come next)
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Pentose Phosphate Pathway Two options for Ribulose-5-P: 1) Ribulose-5-P isomerase

2) Ribulose-5-P epimerase

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Pentose Phosphate Pathway We now have C-5 sugars, but will get C-6 sugars in the end of PPP. How is this done? Transketolase: catalyzes the transfer of C-2 units

The products from previous steps


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Pentose Phosphate Pathway Transaldolase: catalyzes the transfer of C-3 units

The products from previous steps

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Pentose Phosphate Pathway

Again transketolase: catalyzes the transfer of a C-2 unit

From step 5

Previous step

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Pentose Phosphate Pathway

In the Pentose Phosphate Pathway three C-5 sugars are converted to two C-6 and one C-3 sugars.

C5 + C5 C3 + C7 C3 + C7 C6 + C4

(Transketolase) (Transaldolase)

C5 + C4 C6 + C3 (Transketolase) ____________________________ 3 C5 2 C6 + C3 (Overall)

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Glucose-6-phosphate may be regenerated from either the: 3-C glyceraldehyde-3-phosphate or the 6-C fructose-6-phosphate

via enzymes of Gluconeogenesis.

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When in the PPP needed? depends on the metabolic needs of the cell

ribose-5-phosphate, NADPH, and ATP

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When in the PPP needed? depends on the metabolic needs of the cell For nucleotide synthesis: 1) Ribose-5-P is needed for nucleotide synthesis 2) Some NADPH is needed to reduce ribonucleotides to deoxyribonucleotides

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When in the PPP needed? depends on the metabolic needs of the cell

When reducing power is needed for fatty acid or steroid synthesis: Generates a lot of NADPH and G-6-P is regenerated to go back into the PPP, and make more NADPH

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When in the PPP needed? depends on the metabolic needs of the cell When only moderate levels of NADPH are needed: The cycle is used to make some energy. The product of PPP: F-6-P and G-3-P are further used by the glycolytic and citric acid pathways to make ATP

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Glutathione
Glutathione is the most abundant antioxidant in the body. It is a tripeptide, made up of cysteine, glutamic acid, and glycine. Glutathione is special in that it is fat soluble and water soluble, therefore it has anti-oxidant activity throughout the entire body. Being an anti-oxidant, glutathione quenches free radicals, thereby protecting healthy cells from damage. Glutathione also acts as a chelator to bind and escort out of the body heavy metals that cause the free radical damage. Glutathione is an important part of the livers detoxification system so it helps to remove metabolic waste. It also has an important role in transporting amino acids into cells

Erythrocytes use glutathione to eliminate H2O2 and hydroperoxides that can damage hemoglobin or the cell membrane. Hydroperoxides, arising spontaneously in the oxygen-rich environment in red blood cells, react with double bonds in fatty acids of membrane lipids, making membranes leaky.
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Glutathione
Glutathione is present in most cells and protects against oxidative stress, and keeps thiol groups of proteins in a reduced state:

Peroxides are eliminated by glutathione peroxidase:

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Defects in the PPP pathway Glucose-6-Phosphate Dehydrogenase deficiency

Regeneration of reduced glutathione requires NADPH produced in the Pentose Phosphate Pathway.
Reduced glutathione is generated by glutathione reductase:

Erythrocytes need a steady supply of NADPH

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Defects in the PPP pathway

Glucose-6-Phosphate Dehydrogenase deficiency

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Defects in the PPP pathway Glucose-6-Phosphate Dehydrogenase deficiency People who have this deficiency (decreased activity of the enzyme) are sensitive to oxidative damage, which can be seen as hemolytic anemia

Hemoglobin is seen on one side of the cell.

These people if they take anti-malaria drug primaquine or eat fava beans ),( which stimulate peroxide formation, will need high levels of NADPH that the mutant cell cannot supply. Like in sickle-cell anemia, in this deficiency the erythrocytes are resistant to infection by the malaria parasite.
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Defects in the PPP pathway Thiamine (B1) is part of the coenzyme TPP (thiamin pyrophosphate). It promotes conversion of pyruvate to acetyl CoA by removing one carbon Similar reaction in TCA cycle And in transketolase (PPP)

Wernicke-Korsakoff Syndrome Mental disorder due to thiamine deficiency loss of memory, partial paralysis. Particularly in alcoholics that lack this in their diet. Due to a mutated transketolase that has 10-fold reduced affinity to its cofactor thiamine (TPP), the PPP is slowed down. Normally if TPP levels drop this does not affect the enzyme. But in people with the abnormal enzyme this causes disease.

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Defects in the PPP pathway Beriberi Thiamine deficiency

Characterized by: loss of sensation in hands and feet muscular weakness advancing paralysis abnormal heart action

Found mainly in people from Asian countries eat white rice where the husk () has been removed that part has most of thiamine.

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The major pathways of fuel metabolism in mammals Most tissues cannot carry out all the following reactions.

180

Organ specialization

Brain

High respiration rate: responsible for 20% of body oxygen consumption (2% of body mass) High energy requirement about 15% of total energy Most energy is required for the plasma membrane (Na/K)-ATPase needed for maintaining the membrane potential required for nerve pulses. Glucose is the main fuel of the brain (120 gr/day) . During fasting it uses ketone bodies (= products of breakdown of fatty acids) Since there is not much glycogen storage in the brain it requires a constant supply of glucose. Low glucose half of normal 4-5mM results in brain dysfuction. Lower coma and irreversible damage, death.
181

Organ specialization

Muscle Fuels: 1) Glucose from glycogen 2) Fatty acids 3) Ketone bodies During rest the muscle generates glycogen storage. Muscle stores about from total glycogen in the body Glycogen is converted to G-6-P for glycolysis. Muscle cells do not have G-6phosphatase so cannot export glucose. Muscle does not have enzymes for gluconeogenesis. Muscle carbohydrate metabolism serves only the muscle.

182

Organ specialization

Muscle

Muscle contraction
Muscle contraction requires ATP. Most ATP in the body comes from TCA and oxidative phosphorylation. At rest the muscles use 30% of body oxygen. During high exertion it gets ATP from: phosphocreatine + ADP creatine + ATP

This is good for 4 seconds, then the muscle shifts to glycolysis of G-6-P. The flux is too much for TCA and ox. phos., therefore G-6-P is degraded to lactate (anaerobic).
Lactate is released to blood and converted in liver to glucose (Cori cycle)

After 20 sec there is muscle fatigue due to drop of pH by lactate.


183

Organ specialization

Heart

A muscle that acts continuously relies only on aerobic metaboilsm and has many mitochondria ( up to 40% of the cytoplasm).

Contains little glycogen and stored lipids and small amounts of creatine phopshate
During resting it uses fatty acids. During heavy work needs glucose and uses up its supply of glycogen. Therefore the heart needs constant supply of oxygen and fuels from the blood.

184

Organ specialization

Adipose tissue

This tissue must store or release fatty acids as needed. Found mostly under the skin, abdomen, skeletal muscle. A 70 kg person has 15 kg of fat = 590,000 kJ When intracellular glucose levels are low, the hormone sensitive lipase causes fatty acids to be sent into the blood. When glucose levels are OK, there is resynthesis of triglycerols from fatty acids.

185

Organ specialization

Liver The center for keeping levels of circulating fuels, for the brain, muscles and other tissues.

Liver acts as a blood glucose buffer

Uptake and release of glucose in response to hormones and blood glucose levels. After carbohydrate dinner: blood glucose is 6mM glucose converted to G-6-P by glucokinase (isoenzyme of hexokinase). More glucose more enzyme activity, so when all tissues are satisfied it will uptake glucose from blood. (Normal blood glucose: 4 to 8 mM (70 to 150 mg/dL)
186

Organ specialization

Liver Fates of G-6-P in liver: 1) When blood glucose drops under 5mM is converted to glucose by G-6-phosphatase, and travels by blood to organs. During exercise or fasting, low blood glucose causes pancreas to secrete glucagon that acts on liver cells that breakdown glycogen. 2) Converted to glycogen when there is no demand for glucose. 3) Converted to acetyl-CoA via glycolysis and can go to: TCA / fatty acid generation / phospholipids / cholesterol 4) Degraded by the PPP to generate NADPH required for fatty acid synthesis.

187

Organ specialization

Kidney
Filters waste and urea and retains metabolites like glucose. Maintains blood pH by exerting excess H+. It exerts protons like NH4+ (ammonia derived from glutamine or glutamate). The remaining amino acid skeleton: -ketoglutarate can be converted to glucose by the citric acid cycle and then gluconeogenesis. (Only done by liver and kidney), or oxidized by ox. phos. to CO2. Blood In erythrocytes (99% of blood cells) glycolysis is the only pathway since they have no mitochondria and therefore depend of anaerobic glycolysis.

188

Organ specialization

189

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191

Disturbances of metabolism Diabetes Mellitus Third leading cause of death in the USA.

Problem: 1) Insulin is not secreted in efficient amounts, or 2) Insulin does not efficiently stimulate target cells
Results Blood glucose levels are high = hyperglycemia (spill into urine) but cells are still starved for glucose Other pathways become accelerated: gluconeogenesis, triacylglycerol hydrolysis, fatty acid oxidation, and ketone body formation (ketosis) Ketone bodies are acids the body works hard to control blood pH by removing H+, but also other ions are excreted (Na+, K+ ,H2O) and this causes dehydration, and decrease in blood volume.

192

Disturbances of metabolism

Diabetes Mellitus

Type I: Insulin-dependent or juvenile-onset diabetes The pancreas lacks or has defective cells (responsible for insulin secretion) Usually an autoimmune response that destroys the cells, noticed after 80% of cells have been eliminated.

Occurs usually in childhood. Daily injections of insulin and balance diet increase the survival, but still there can be many organ complications (kidney, nerves, cardiovascular, cataract)
Successful treatment: cell transplantation

Pancreatic lesion

193

Disturbances of metabolism

Diabetes Mellitus

Type II: (Non-insulin dependent or maturity-onset diabetes)


90% of diagnosed diabetes cases, usually occurs in obese people with genetic disposition. Normal or elevated insulin levels but cells do not respond to insulin = insulin resistant. Why? In few cases there is a mutation in the insulin receptor. In most cases a genetic cause has not been found. Probably due to overeating that causes increased insulin production.

The hyperglycemia induces more insulin production by the cells


Diet alone can decrease the degree of the disease. There are drugs that decrease insulin resistance by decreasing glucose release by the liver or increasing glucose disposal in muscle.
194

Disturbances of metabolism Obesity

However, in most obese humans leptin levels increase when body fat rises. The defect is probably not in leptin production but in leptin resistance = decrease in the levels of the leptin receptor in the brain (similar to diabetes type II) Also low response to leptin increases neuropeptide Y which in the brain stimulates appetite. There are also other hormones that are involved in obesity.

195

Hormonal control of metabolism In animals coordination of activities between organs is done by hormones. Hormones are synthesized by endocrine glands.

They function in:

1) Maintain homeostasis steady state of blood glucose levels - insulin


2) Respond to external stimuli epinephrine 3) Developmental programs sexual differentiation and maturation, pregnancy

196

196

Hormonal control of metabolism Islets of Langerhans endocrine cell that secrete polypeptides into the blood stream cells insulin, in response to high glucose cells glucagon, in response to low glucose

Pancreatic hormones

197

197

Insulin

Pancreatic cells are most sensitive to blood glucose at levels of 5.5 6.0 mM (normal is 3.6-5.8 mM)
Glucose diffuses into the cells and is sensed by the sensor glucokinase (like liver) In these cells all G6P is converted to: pyruvate citric acid cycle oxidative phospohrylation (No glycogen storage, low PPP, low lactate dehydrogenase)

Mechanism is not known but oxidative phosphorylation levels regulate insulin synthesis and secretion.

198

198

Insulin

Glucose transporter Glut4

Insulin promotes glucose uptake in muscle and adipose tissue and inhibits hepatic glucose production.

The Glut4 system allows the tissues to quickly accumulate glucose after a meal.

Once glucose enters cells it can be used for synthesizing: Glycogen in muscle Triacylglycerols in adipocytes Insulin promotes these activities.

199

199

Insulin

Glucose transporter Glut4

Muscle cells and adipocytes

200

200

Insulin In liver cells - insulin does not increase rate of glucose uptake. Instead it:

Decreases levels of glycogenolysis Promotes glycogen synthesis


Inhibits transcription of genes for gluconegenesis Stimulates transcription of genes of glycolysis

Results: Liver stores glucose as glycogen and triacylglycerols (instead of making glucose by glycogenolysis and gluconeogenesis)

201

201

Glucagon Leads to glycogenolysis (glycogen breakdown) in the liver

Makes glucose available to other tissues when glucose levels drop

Muscle cells do not respond to glucagon dont have a receptor, therefore get the glucose indirectly from the liver

202

202

Hormonal control of metabolism

Adrenal hormones

The adrenal glands release hormones in response to neuronal signals. The cortex synthesizes and secretes steroid hormones The medulla synthesizes epinephrine and nor-epinephrine They are released during stress.

203

203

Epinephrine Liver Epinephrine promotes the release of glucagon from the pancreas in liver it stimulates glycogen breakdown Epinephrine binds to receptors on liver cells (adrenergic receptors) and turns on cAMP signal transduction, leading to glycogenolysis and gluconeogenesis

Muscle

Epinephrine stimulates glycogenolysis glucose glycolysis ATP


Adipose tissue Causes breakdown of fat to fatty acids for use in other tissues
204

204

Glycogen If this much glucose were dissolved in the cytosol of a hepatocyte, its concentration would be about 0.4 M, and will cause osmotic problems in the cell. When stored as a long polymer (glycogen), however, the same mass of glucose has a concentration of only 0.01 M. Glycogen granules are complex aggregates of glycogen and the enzymes that synthesize it and degrade it, as well as the machinery for regulating these enzymes.

The elementary particle of glycogen consists of up to 55,000 glucose residues


Glycogen granules contain the enzymes for glycogen metabolism

205

206

206

207

207

208

208

Actions of glucagon in liver that lead to a rise in blood glucose.

209

Glycogen Glucose is essential for brain and red blood cells, that depend entirely on glucose. When glucose is required one source is glycogen. Glycogen is stored mainly in liver & skeletal muscle. 10% of the weight of liver and 1% to 2% of the weight of muscle. Glycogen from diet meat. Glycogen storage is way for higher organisms to protect themselves from fuel shortage.

210

Glycogen

Why does the body store glucose as glycogen, when it could use fat that is more abundant in the body? 1( Muscles cant use fat as rapidly as they can glycogen 2) Fatty acids (from fat) cannot be used anaerobically 3) Animals cannot turn fatty acids into glucose

211

Glycogenolysis = glycogen breakdown Glucose is removed from ends of branches - two possible reactions: Hydrolysis: usually for digested glycogen (food). In this reaction elements of water are added to glycosidic bond. Hydrolysis yields glucose that can be absorbed in blood. Phosphorolysis: usually for stored glycogen. In this reaction elements of phosphoric acid are added to glycosidic bond. The advantage is a sugar phosphate that can be utilized directly in the glucose pathways within the cells.

212

Glycogenolysis

1. Glycogen phosphorylase

Pyridoxal phosphate (PLP), a derivative of vitamin B6, serves as prosthetic group for Glycogen Phosphorylase.

Lys

It is held at the active site on a lysine residue.

In contrast to the role of this cofactor in other enzymes, the phosphate of PLP is involved in the catalytic activity of the enzyme.

213

Glycogenolysis 1. Glycogen phosphorylase

PLP
A class of drugs developed for treating the hyperglycemia of diabetes (chloroindolecarboxamides), inhibit liver Phosphorylase allosterically. These inhibitors bind at the dimer interface, stabilizing the inactive (tense) conformation.

GlcNAc

inhibitor

GlcNAc Human Liver Glycogen Phosphorylase

PLP

PDB 1EM6

Question: Why would an inhibitor of Glycogen Phosphorylase be a suitable treatment for diabetes?
214

Glycogenolysis

1. Glycogen phosphorylase Releases glucose only if is 5 units away from branch point Phosphorolytic cleavage of the a(14) glycosidic linkages

215

Glycogenolysis

1. Glycogen phosphorylase

A dimer of two identical subunits of 831 amino acids


Has an active site for the phosphorolysis of glycogen, and a glycogen binding site.

Binds a cofactor: PLP (pyridoxal-5-phosphate) Has an: Active state (R = relaxed) Inactive state (T = tense) in which the active site is buried

Glycogen binding site probably allows the enzyme to work many times on the same molecule
216

Glycogenolysis 2. Glycogen debranching enzyme

Phosphorolysis leaves a limit branch of 45 residues from branch point


The enzyme is a (1,41,4) transglycosylase and has 2 different catalytic sites. The transferase of the debranching enzyme transfers 3 glucose residues from to the end of another branch, diminishing the limit branch to a single glucose.

The (16) glucosidase then catalyzes hydrolysis of the (16) linkage, yielding a free glucose.
The major product of glycogen breakdown is glucose-1-phosphate, from Phosphorylase activity.
217

Glycogenolysis 2. Glycogen debranching enzyme

218

Glycogenolysis 3. Phosphoglucomutase G-1-P is catalyzed to G-6-P. Reversible and can lead to glycogen synthesis. In muscle G-6-P coming from glycogen can enter glycolysis and supply energy. Luck of G-6-Phosphatase insures that glucose-6-P can not diffuse out of the cells, therefore it can only be catabolized in glycolysis In liver it can be dephosphorylated and released to the blood. This requires a specific liver enzyme: G-6-P phosphatase.

219

Glycogenolysis 3. Phosphoglucomutase In the liver, kidney and intestine )other cells dont have G-6-phosphatase)

220

Glycogen

Glucose-1-P

Glucose Hexokinase or Glucokinase Glucose-6-Pase Glucose-6-P Glucose + Pi Glycolysis Pathway

Pyruvate Glucose metabolism in liver.


The reversible Phosphoglucomutase may also convert glucose-6phosphate glucose-1-phosphate, precursor for glycogen synthesis as well as glycogen breakdown product.
221

Metabolic Disorders Congenital a) gene is absent b) under expressed c) poorer functioning mutant Acquired a) disease of organ b) disease of endocrine organ

222

Glycogen storage diseases

Glycogen storage diseases are congenital genetic (inherited) enzyme deficiencies that result in excessive glycogen accumulation within cells.

Additional symptoms depend on the particular enzyme that is deficient.

They cause abnormal quantity or quality of glycogen. Result in: Liver: hepatomegaly (enlarged liver) and hypoglycemia (low blood sugar)

Muscles: cramps and weakness

229

Glycogen storage diseases Type I: Glucose-6-phosphatase deficiency )von Gierkes disease( Increase in cellular G-6-P and accumulation of glycogen in liver and kidney (G-6-P inhibits glycogen phosphorylase). No glycogen breakdown and gluconeogenesis. Inability to increase blood sugar levels in response to glucagon Hepatomegaly, hypoglycemia, small stature (1 in 100,000)

Normal

Treatment to increase blood glucose : * Inhibition of liver glucose uptake (drugs) to increase blood levels * Intragastric feeding overnight to increase blood levels Liver filled * Oral feeding with corn starch that breaks with down slowly to glucose glycogen or *Liver transplantation * or Gene therapy G6Pase knockout mice were treated with a viral vector containing 230 G6Pase - increases survival rate Survival to adult was rare, now typical

Glycogen storage diseases Type V: Muscle phosphorylase deficiency )McArdles disease( Symptoms: Painful muscle cramps Appear at early adulthood Glycogen breakdown system cannot provide enough fuel for glycolysis when there is high demand for ATP (Liver phosphorylase is OK) Prevented by avoiding strenuous exercise Treatment: oral B6 vitamin (PLP) (1 in 100,000)

231

Glycogen storage diseases Type VI: Liver phosphorylase deficiency )Hers disease(

Similar symptoms to those with Type I glycogen storage disease Hypoglycemia and hepatomegaly Hypoglycemia because of the inability of glycogen phosphorylase to respond to need for glucose production by the liver

Stunted growth

Good prognosis

232

Control of glycogen breakdown: Commonly used terminology: "a" is the form of the enzyme that tends to be active, and independent of allosteric regulators (in the case of Glycogen Phosphorylase, when phosphorylated). "b" is the form of the enzyme that is dependent on local allosteric controls (in the case of Glycogen Phosphorylase when dephosphorylated).

233

Control of glycogen breakdown:

Allosteric regulation

Glycogen Phosphorylase b in muscle is subject to allosteric regulation:


AMP (present significantly when ATP is depleted) activates phosphorylase, promoting the relaxed conformation. ATP & glucose-6-phosphate, which both have binding sites that overlap that of AMP, inhibit phosphorylase, promoting the tense conformation. glycogen breakdown is inhibited when ATP and glucose-6-phosphate are plentiful.

234

Control of glycogen breakdown: When blood glucose levels are low

Control by phosphorylation

cAMP cascades are activated by the hormones

Glucagon- liver (secreted form the pancreas)


Epinephrine muscle (secreted from the adrenal)

Both hormones are produced in response to low blood glucose. )Epinephrine )adrenaline( is also part of the fight or flight response. (Acute stress response. Reaction of an animal to threats through the nervous system, priming the animal for fighting or fleeing.)

235

Control of glycogen breakdown:

Control by phosphorylation

Hormone (glucagon or epinephrine) released in response to metabolic state Synthesis of cAMP from ATP by adenylate cyclase P* Activation of protein kinase A (cAMP dependent protein kinase) P*

Activation of phosphorylase b kinase


P* Activation of glycogen phosphorylase

Glycogen breakdown

236

Control of glycogen breakdown:

Control by phosphorylation

The cAMP cascade results in phosphorylation of a serine hydroxyl of Glycogen Phosphorylase, which promotes transition to the active (relaxed) state glycogen phosphorelase a. The phosphorylated enzyme is less sensitive to allosteric inhibitors. even if cellular ATP & glucose-6-phosphate are high, Phosphorylase will be active. The glucose-1-phosphate produced from glycogen in liver may be converted to free glucose for release to the blood. With this hormone-activated regulation, the needs of the organism (hormone) take precedence over needs of the cell (cellular ATP).

237

Control of glycogen breakdown:

Control by phosphorylation

In a resting cell: ATP and G-6-P are usually high enough inhibit phosphorylase b (mostly in T state) Activity of phosphorylase in a resting cell depends on how much is in the phos. a state this depends on how much phosphorylation there is and on the levels of glucose. If glucose is high insulin secretion dephosphorylation it will be in an inactive form (T)

238

Control of glycogen breakdown: cAMP Glucagon epinephrine


activates

Control by phosphorylation

239

Insulin

Control of glycogen breakdown: Control by phosphorylation Cascade mechanism of epinephrine and glucagon action. The resulting breakdown of glycogen provides glucose, which in the myocyte can supply ATP (via glycolysis) for muscle contraction and in the hepatocyte is released into the blood to counter the low blood glucose. Note the amplification of the signal: from few molecules of hormone to thousands of glucose molecules

240

Glycogen synthesis Glycogen synthesis and glycogen breakdown are separate pathways. McArdles disease )Muscle phosphorylase deficiency) showed that glycogen breakdown in muscle is deficient due to lack of glycogen phosphorylase, but glycogen levels were moderately high indicating that phosphorylase was not required for synthesis. Key molecule is UDP-glucose (uridine-diphosphate glucose)

241

Glycogen synthesis

UDP-glucose-pyrophosphorylase

The cell makes a high energy molecule UDP-glucose from G-1-P

Why glucose-1-P needs to be converted to sugar-nucleotide?


1. The reaction is irreversible this means that once formed, UDPglucose has to be incorporated to glycogen 2. High energy bond provides free energy for the enzymatic reaction

3. UDP is good leaving group thus promoting enzymatic reaction


4. By marking hexoses in different ways, cell can separate processes

242

Glycogen synthesis
2Pi

243

Glycogen synthesis The reactions are: 1) glucose-1-phosphate + UTP UDP-glucose + PPi 2) PPi + H2O 2 Pi

Overall: glucose-1-phosphate + UTP UDP-glucose + 2 Pi


The spontaneous hydrolysis of the ~P bond in PPi (P~P) drives the overall reaction.

244

Glycogen synthesis Branching enzyme

Transfers a segment from the end of a glycogen chain to the C6 hydroxyl of a glucose residue of glycogen to yield a branch with an (16) linkage. About 7 glucoses are transferred Branching process increases glycogen solubility and increases a number of non-reducing ends (important for rapid glycogen breakdown)

245

Glycogen synthesis

246

247

Glycogen Breakdown and Synthesis Are Reciprocally Regulated

If both pathways would work at the same time we would have a futile cycle

Glycogen Synthesis UTP UDP + 2 P i glycogen(n) + glucose-1-P Glycogen Phosphorylase glycogen(n + 1) Pi

Allosteric & phosphorylation control

248

Control of glycogen synthesis:

Allosteric regulation

Glycogen Synthase is allosterically activated by: glucose-6-phosphate (opposite of the effect on Phosphorylase). glycogen synthesis is activated when glucose-6-phosphate is plentiful. These controls benefit the cell because it is more useful to a cell to store glucose as glycogen when there is enough: input to Glycolysis (glucose-6-phosphate) main product of Glycolysis (ATP)

249

Control of glycogen synthesis: Control by phosphorylation Glycogen synthase a active (dephosphorylated) Glycogen synthase b less active (phosphorylated) The cAMP cascade induced in liver by glucagon or epinephrine has the opposite effect on glycogen synthesis = inhibition Glycogen Synthase is directly phosphorylated by cAMP-Dependent Protein Kinase, as well as by Phosphorylase Kinase. Phosphorylation of Glycogen Synthase promotes the "b" (less active) conformation.

250

Hormonal effects

Insulin antagonizes the effects of the cAMP cascade induced by glucagon & epinephrine.
Insulin is produced in response to high blood glucose

251

Hormonal effects Insulin, produced in response to high blood glucose, triggers a separate signal cascade that leads to activation of Phosphoprotein Phosphatase. This phosphatase catalyzes removal of regulatory phosphate residues from Phosphorylase, Phosphorylase Kinase, & Glycogen Synthase enzymes. Thus insulin antagonizes effects of the cAMP cascade induced by glucagon & epinephrine.

252

Hormonal effects Ca++ also regulates glycogen breakdown in muscle. During activation of contraction in skeletal muscle = nerve pulse, the sarcoplasmic reticulum (smooth ER of muscle) releases Ca++. Ca++ released to the cytosol activates actin/myosin interactions and glycogen breakdown (for glycolysis ATP muscle contraction).

253

Hormonal effects

Phosphorylase Kinase Phosphorylase Kinase-Ca++ P-Phosphorylase Kinase-Ca++

inactive partly active fully active

Phosphorylase Kinase in muscle includes calmodulin (Calcium-modulating protein) as its subunit. Phosphorylase Kinase is partly activated by binding of Ca++ to this subunit. Calmodulin binds 4 Ca++ ions and the enzyme undergoes a conformational change. Phosphorylation of the enzyme, via a cAMP cascade induced by epinephrine, results in further activation. These regulatory processes ensure release of phosphorylated glucose from glycogen, for entry into glycolysis to provide ATP needed for muscle contraction.
254

Hormonal effects Liver: Insulin and glucagon are both made in the pancreas respond to blood glucose levels Muscles and other tissues: Insulin and epinephrine (adrenaline) and nor-epinephrine affect glucose metabolism

Epinephrine induces glycogen breakdown to G-6-P in muscle for glycolysis, or to glucose for export from liver. When there is enough glucose, insulin stimulates glucose uptake by muscle cells and increases glycogen synthesis. Liver cells respond directly to glucose and increase glycogen synthesis. 255

Hormonal effects

Difference in the regulation of carbohydrate metabolism in liver and muscle. In liver, either glucagon (indicating low blood glucose) or epinephrine (signaling the need to fight or flee) has the effect of maximizing the output of glucose into the bloodstream.

256

In muscle, epinephrine increases glycogen breakdown and glycolysis, which together provide fuel to produce the ATP needed for muscle contraction.

Glycogen phosphorylase b
Glycogen synthase a
ATP

epinephrine

+
- " phosphoprotein phosphatase
- " phosphoprotein phosphatase " protein kinase

+
" protein kinase

+
Ca+2 AMP

Glycogen phoshorylase a Glycogen synthase b


257

P ATP

Glycogen phosphorylase b

Glycogen synthase a
glucagon

+
- " phosphoprotein phosphatase
- " phosphoprotein phosphatase " phosphorelase b kinase

" protein kinase

Glycogen phoshorylase a

Glycogen synthase b
258

Glycogen storage diseases Type IV: Branching enzyme deficiency )Andersens disease( Most severe dont live to more than 5 years Type IV glycogen is not increased in liver but the structure is abnormal Very long unbranched glycogen decreases solubility, liver failure

259

Gluconeogensis

260

261

262

Tissues that synthesize and use glucose

& dont have the machinery to synthesize it


263

Humans consume 160 g of glucose per day 75% of that is in the brain (120 g) Body fluids contain only 20 g of glucose Glycogen reserves yield 180-200 g of glucose The body must be able to make its own glucose when dietary sources of glucose are not available or when the supply of liver glycogen is exhausted Gluconeogenesis In the liver, and less in the kidney

Synthesis of "new glucose" from common metabolites: pyruvate, lactate, glycerol, amino acids and all TCA intermediates

Biosynthesis of a carbohydrate from 3-carbon and 4-carbon precursors

264

Gluconeogenesis

The brain and erythrocytes are almost completely dependent on glucose as an energy source.
The glycogen stores in the liver will supply the brain with glucose for half a day of fasting or starvation. When fasting most of the bodys glucose needs must be supplied by gluconeogenesis. Was shown that during a fast: During the first 22 hours 64% of glucose come from gluconeogenesis By 46 hours 100% is from gluconeogenesis Gluconeogenesis is an universal passway and it exists in all organisms

265

Gluconeogenesis

Non-carbohydrate precursors: Lactate, pyruvate from glycolysis in skeletal muscle or erythrocytes Amino acids from dietary proteins, or muscle protein after starvation Glycerol from fat breakdown and others Acetyl-coA is not a precursor for gluconeogenesis

266

Gluconeogenesis

Synthesis of glucose from pyruvate utilizes many of the same enzymes as glycolysis. Three glycolysis reactions have such a large negative Gs that they are essentially irreversible. These must be bypassed in gluconeogenesis: Hexokinase Phosphofructokinase Pyruvate Kinase.

Therefore gluconeogenesis requires 4 specific enzymes. The other steps use reversible glycolytic enzymes.

267

Gluconeogenesis

Glycolysis

268

Gluconeogenesis

Glycolysis

269

Gluconeogenesis

Need to bypass: pyruvate kinase (pyrPEP) PEP has a higher negative G of phosphate hydrolysis than ATP, so will have to break 2 phosphate bonds to get to PEP. Pyruvate to phosphoenolpyruvate
A. Pyruvate carboxylase

B. Phosphoenolpyruvate carboxykinase (PEPCK)

270

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase

Requires ATP and biotin (B7) Oxaloacetate is a highenergy intermediate

271

Gluconeogenesis

Pyruvate to phosphoenolpyruvate
A. Pyruvate carboxylase The enzyme has a biotin prosthetic group. Biotin is an essential nutrient. Biotin functions as a CO2 carrier by forming a carboxyl group at its end. Biotin has a 5-C side chain whose terminal carboxyl is in amide linkage to a lysine amino group in the enzyme. A highly specific enzyme Biotin Protein Ligase catalyzes attachment of biotin to a particular Lys of Pyruvate Carboxylase (and to specific Lys residues in other enzymes that catalyze carboxylation or decarboxylation reactions). The biotin & lysine side chains form a long swinging arm that allows the biotin ring to swing back & forth between 2 active sites (one carboxilates biotin and the other pyruvate)
272

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase 1) Biotin carboxylation is catalyzed at one active site of Pyruvate Carboxylase: ATP is cleaved to ADP and Pi Pi Reacts with bicarbonate HCO3- to release CO2 CO2 is bound to biotin to form an activated carboxyl

273

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase 2) At the other active site of Pyruvate Carboxylase, the activated carboxyl is transferred from carboxybiotin to pyruvate to form oxaloacetate.

274

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase Biotin is an essential nutrient, but deficiency is rare. Avidin, a protein in egg whites with a barrel structure, tightly binds biotin. Excess consumption of raw eggs can cause nutritional deficiency of biotin. The strong avidin-to-biotin affinity is used by biochemists as a specific "glue." To bind proteins together, biotin may be covalently linked to one protein and avidin to the other.

Avidin with bound biotin

275

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase Oxaloacetate is: 1) a precursor for gluconeogenesis But is also: 2) An intermediate of citric acid cycle. When the citric acid cycle is inhibited (there is enough ATP), oxaloacetate is sent to gluconeogenesis But oxaloacetate is made by the citric acid cycle in the mitochondria and the next steps are in the cytosol So OAA must be transported out
276

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase

There is no transport system for oxaloacetate:


Must be converted to malate or aspartate OAA is reduced by malate dehydrogenase For the malate pathway NADH is used in the mitochondria but is then made back in the cytosol. Malate is reduced agent for the NADH synthesis in cytosol. NADH is necessary for gluconeogenesis (glyceraldehyde-3-P dehydrogenase). NADH/NAD+ in cytosol = 8*10-4 NADH/NAD+ in mito :105 times greater

NADH levels are low in the cytosol and the cells needs it for gluconeogeneis to proceed. 277

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase The first partial reaction of pyruvate carboxylase, the formation of carboxybiotin, depends on the presence of acetyl-CoA. Biotin is not carboxylated unless acetylCoA is bound to the enzyme. Pyruvate Carboxylase is allosterically activated by acetyl-CoA. Acetyl-CoA has no effect on the second partial reaction. If acetyl-CoA is high, it signifies the need for more oxaloacetate Then, on the cell level: 1) If the energy charge is high, it means that there are enough substrates for the citric acid cycle, and oxaloacetate is converted into glucose (gluconeogenesis). 2) If the energy charge is low, oxaloacetate replenishes the citric acid cycle.
278

Gluconeogenesis Pyruvate to phosphoenolpyruvate A. Pyruvate carboxylase

If acetyl-CoA is high, it signifies the need for more oxaloacetate

Then, on the cell level: 1) If the energy charge is high, it means that there are enough substrates for the citric acid cycle, and oxaloacetate is converted into glucose (gluconeogenesis). 2) If the energy charge is low, oxaloacetate replenishes the citric acid cycle.
279

Gluconeogenesis Pyruvate to phosphoenolpyruvate B. Phosphoenolpyruvate carboxykinase (PEPCK)

The CO2 is removed


GTP is used

280

Gluconeogenesis Pyruvate to phosphoenolpyruvate B. Phosphoenolpyruvate carboxykinase (PEPCK) The reaction is thought to proceed in two steps: Oxaloacetate is first decarboxylated to yield a pyruvate enolate anion intermediate. This is phosphorylated by phosphate transfer from GTP. A metal ion such as Mn++ is required, in addition to the Mg++ associated with the nucleotide substrate.

281

Gluconeogenesis Pyruvate to phosphoenolpyruvate

A. Pyruvate carboxylase

B. Phosphoenolpyruvate carboxykinase (PEPCK)

282

Gluconeogenesis PEP is converted to Fructose-1,6-bisphosphate using the same glycolytic enzymes

283

Gluconeogenesis Specific enzymes for bypass reactions: Hydrolytic reactions, no ATP Release of Pi in an exergonic process 1) Fructose-1,6-bisphosphate to Fructose-6-phosphate by Fructose-1,6-bisphosphatase 2) Glucose-6-phosphate to Glucose by Glucose-6-phosphatase (used in glycogen breakdown)

284

Gluconeogenesis Gluconeogenesis occurs only in liver and kidney because of the presence of Glucose-6-phosphatase. Only these tissues supply glucose to other tissues. Muscle and brain do not do gluconeogenesis G-6-phosphatase is found in the ER (active site is exposed to cytosol) G-6-P is hydrolyzed as it passes into the ER and glucose enters the ER ER vesicles filled with glucose diffuse to the plasma membrane, fuse with it and open, releasing glucose into the bloodstream.

285

Gluconeogenesis Energy expenses: Anabolic pathways cost energy Glycolysis & Gluconeogenesis are both spontaneous (due to their irreversible steps). Glycolysis: glucose + 2 NAD+ + 2 ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP Gluconeogenesis: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi

286

Gluconeogenesis

If both pathways were simultaneously active in a cell, it would constitute a "futile cycle" that would waste energy.

Glycolysis: glucose + 2 NAD+ + 2 ADP + 2 Pi


2 pyruvate + 2 NADH + 2 ATP Gluconeogenesis: 2 pyruvate + 2 NADH + 4 ATP + 2 GTP glucose + 2 NAD+ + 4 ADP + 2 GDP + 6 Pi

Glycolysis yields 2 ~P. Gluconeogenesis expends 6 ~P.


Futile cycle of both pathways would waste 4 ~P per cycle. To prevent this waste, glycolysis & gluconeogenesis are reciprocally regulated.

287

Regulation of gluconeogenesis Glycolysis and gluconeogenesis are reciprocally controlled Regulation occurs on the strongly exerogenic processes in each pathway the non-reversible enzymes * When glycolysis is turned on gluconeogenesis should be turned off

High energy status: glycolysis off Pyruvate and other substrates are used for synthesis and storage of glucose

Low energy status: glycolysis on, to rapidly degrade glucose and supply energy
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(Gs)

Regulation of gluconeogenesis Allosteric control by adenine nucleotides (ATP/AMP)


Allosteric = the activity of the active site is affected by the binding of a molecule to another part of the enzyme

When cellular ATP is high (low AMP), glucose is not degraded to make ATP. Under such conditions the cell prefers to store glucose as glycogen. Phosphofructokinase (Glycolysis) is inhibited by ATP and stimulated by AMP. Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP. ATP regulates pyruvate kinase in liver cells: If ATP is high it inhibits glycolysis and induces gluconeogenesis that occurs in liver.

289

Regulation of gluconeogenesis

290

Regulation of gluconeogenesis

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Regulation of gluconeogenesis

Glucose-6-phosphatase is under substrate-level control, not allosteric control The Km of the enzyme glucose-6 phophatase is higher than intracellular levels of G6P, and will then work when the levels are high.

(Hexokinase, as we learned, is inhibited by G-6-P but in an allosteric manner)

292

Regulation of gluconeogenesis Allosteric control Fructose-2,6-bisphosphate

Fructose-2,6-bisphosphate is not a glycolytic intermediate but controls both pathways. F-2,6-bisphosphate: 1) a potent activator of PFK (PFK1) from glycolysis 2) an inhibitor of Fructose 1,6 BPase from gluconeogenesis F-2,6-bisphosphate is synthesized and degraded by the same molecule with two enzymatic activities: PFK2 and fructose bisphosphatase-2

293

Regulation of gluconeogenesis

Allosteric control Fructose-2,6-bisphosphate

Global control in liver cells includes reciprocal effects of a cAMP cascade, induced by glucagon when blood glucose is low. Phosphorylation of enzymes by cAMP-Dependent Protein Kinase results in inhibition of Glycolysis & stimulation of Gluconeogenesis, making glucose available for release to the blood.

FBase is stimulated (release of inhibition by F2,6BP)

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Pyruvate Kinase (Glycolysis) is also inhibited when phosphorylated via cAMP-Dependent Protein Kinase

Regulation of gluconeogenesis

Allosteric control Fructose-2,6-bisphosphate

Phosphofructokinase (PFK-1) is normally activated by fructose-2,6-bisP, so a cAMP-induced decrease in [fructose-2,6-bisP] slows this Glycolysis enzyme.
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Glycogen
X

Pyruvate Gluconeogenesis Glucose-6-P Glucose + Pi Glucose-6-Pase


X

Glucose-1-P

Glycolysis Pathway
Summary of effects of glucagon-cAMP cascade in liver: Gluconeogenesis is stimulated. Glycolysis is inhibited. Glycogen breakdown is stimulated. Glycogen synthesis is inhibited. Free glucose is formed for release to the blood.
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Regulation of gluconeogenesis

The Cori Cycle

The Cori Cycle connects between muscle and liver It operates during exercise, when aerobic metabolism in muscle cannot keep up with energy needs.

For a brief burst of ATP utilization (2 to 7 sec), muscle cells utilize ~P stored as phosphocreatine. For more extended exercise, ATP is mainly provided by Glycolysis.
In order to avoid a futile cycle in one cell glycolysis occurs in muscle and gluconeogeneis in the liver

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Regulation of gluconeogenesis

The Cori Cycle

Vigorous exercise can lead to a buildup of lactate and NADH, due to oxygen shortage and the need for more glycolysis NADH can be reoxidized during the reduction of pyruvate to lactate Lactate is then returned to the liver through the bloodstream, where it can be reoxidized to pyruvate by liver Lactate Dehydrogenase

Pyruvate is then turned into glucose by gluconeogenesis


298

299

The Cori cycle costs 6 ~P in liver for every 2 ~P made available in muscle. The net cost is 4 ~P. Although costly in ~P bonds, the Cori Cycle allows the organism to accommodate to large fluctuations in energy needs of skeletal muscle between rest and exercise.
300

Gluconeogenesis Pyruvate to phosphoenolpyruvate There is another bypass from pyruvate to PEP when lactate is the glucogenic precursor. When lactate dominates (after exercise) the conversion of lactate to pyruvate in the cytosol of hepatocytes yields NADH, and the export of reducing equivalents (as malate) from mitochondria is therefore unnecessary.

The oxaloacetate is then converted directly to PEP by a mitochondrial isozyme of PEP carboxykinase, and the PEP is transported out of the mitochondrion to continue on the gluconeogenic path.

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Outline of pathways for glucose synthesis from the major gluconeogenic precursors.

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Gluconeogenesis Major gluconeogenic precursors amino acids

Red - glucogenic amino acids Yellow ketogenic amino acids

During starvation breakdown of muscular proteins is a major source for gluconeogenesis


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Gluconeogenesis Major gluconeogenic precursors glycerol TG breakdown yields fatty acids and glycerol. Glycerol can be used in gluconeogenesis converts to glycerol-3-P first and then to DHAP.

Fatty acids oxidation yields acetyl-coA not a gluconeogenetic precursor (unless there is glyoxylate cycle)

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Animations

http://www.wiley.com/legacy/college/boyer/0470003790/animations/gluconeoge nesis/gluconeogenesis.swf

http://www.wiley.com/legacy/college/boyer/0470003790/animations/cori_cycle/cori_c ycle.htm

http://wps.prenhall.com/wps/media/objects/2398/2456197/AACLRQZ0.mov http://wps.prenhall.com/wps/media/objects/2398/2456197/AACLRQZ0.mov

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Hb A1C () 2-3 . . , . , . , , 3 .

Phenylketonuria examination

Lipids, Membranes, and Cellular Transport , , - ( ) () : .Golgi, ER, Mitochondria ' '. . , . , : . ( , ). ( )E .

Vitamin D

Vitamin E

Vitamin K
K1

K2

Vitamin C

GOUT

Uric acid

Excretion of bilirubin

386

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