bio chem 2

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Medical BiochemistryMedical Biochemistry

Review #2Review #2

ByByJason ElmerJason Elmer

[email protected]@uic.edu

Obi EkwennaObi Ekwenna

[email protected]@uic.edu

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YOUR EXAMYOUR EXAM

Lectures 14-24 ~44 questions (4 questions per lecture)

Take a calculator to the exam

Exam on Monday October 4th.

DO THE STUDY QUESTIONS; if nothing

else read the answers!!!!!!!!!!

Of course TLEs are highly recommended!

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It is impossible to memorizeIt is impossible to memorize

every possible bit ofevery possible bit ofbiochemistry trivia. Theybiochemistry trivia. They

simply know way too muchsimply know way too muchabout metabolism for a singleabout metabolism for a single

person to be able toperson to be able to

regurgitate it all.regurgitate it all.

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Do not rely on passive reading and

highlighting/underlining of the textbook.

Do not sit and stare at the handouts Do not try to read 50 review books. (Make

your own review book instead!)

Do focus on identifying key concepts Do actively draw and redraw pathways

and connections

Do learn to identify relevant information

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Do prioritize:

What is the purpose of a pathway?

What are the starting and ending molecules?

Where is the pathway (in the cell, in a tissue, in an organ system)?

How does the pathway connect to other pathways?

What metabolic conditions turn the pathway on and off?

What are the control points for regulating the pathway?

reactants, products and enzyme name of each regulatory step additional regulatory molecules involved (vitamins, cofactors)

make sure you know every step that makes or uses ATP

What structural features are important for the function and interactionof specific regulatory molecules in a pathway?

What biochemical techniques are used to study these pathways? What specific drugs or diseases associated with the pathway?

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METABOLIC PATHWAYSMETABOLIC PATHWAYS

Glycolysis

Gluconeogenesis

Citric Acid Cycle (Krebs Cycle) Glycogen Metabolism

Hexose Interconversions

Electron Transport Chain

Oxidative Phosphorylation

Pentose-Phosphate Shunt

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GLYCOLYSISGLYCOLYSIS Oxidation of glucose is known as Glycolysis.

EitherAerobicPyruvate Anaerobic Lactic Acid

Occurs in the Cytosol

Overall Rxn:Glucose + 2 ADP + 2 NAD+ + 2 Pi 2 Pyruvate + 2 ATP + 2 NADH + 2 H+

NADH generated during glycolysis is used to fuelmitochondrial ATP synthesis via oxidative phosphorylation.Does not pass through mitochondrial membrane

2 ATP generated glycerol phosphate shuttle

3 ATP generated malate-aspartate shuttleIf used to transport the electrons from cytoplasm NADH into

the mitochondria.

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Key ReactionsKey Reactions

Hexokinase Found in the cytosol of most tissues

Low specificity: its a hoe for hexoses

Low Km: high affinity for glucose

Inhibited by Glucose-6-phosphate

Glucokinase: Found in the Liver and pancreatic bcells

Also a hexokinase

High specificity for glucose

High Km

inhibited by fructose-6-phosphate

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Regulation ofGlycolysisRegulation ofGlycolysis Hexokinase, PFK-1 and PK all proceed with a

relatively large free energy decrease. These non-equilibrium reactions of glycolysis would be idealcandidates for regulation of the flux throughglycolysis.

Hexokinase is not key because of G6P is generatedby glycogenolysis

PK reaction is reversed in Gluconeogenesis

Therefore rate limiting step in glycolysis is thereaction catalyzed by PFK-1.

PFK-1 is a tetrameric enzyme that exist in twoconformational states termed R and T that are inequilibrium.

ATP is both a substrate and an allosteric inhibitor of

PFK-1. F6P is the other substrate for PFK-1 and it also bindspreferentially to the R state enzyme. ATP binds the T state.

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The inhibition of PFK-1 by ATP is overcome by

AMP which binds to the R state of the enzyme

and, therefore, stabilizes the conformation of the

enzyme capable of binding F6P.

The most important allosteric regulatorof both

glycolysis and gluconeogenesis is fructose 2,6-

bisphosphate, F2,6BP, which is not an

intermediate in glycolysis or in gluconeogenesis.

Also important to note that Insulin/Glucagon ratio

i.e. fed/starve state, regulate Pyruvate Kinase

activity. The last enzyme in the pathway. Glucagon: high in starvation, b/cos blood

glucose levels are low, therefore it favors

gluconeogenesis in Liver.

Insulin: on the contrary favors glycolysis.

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GlycolysisGlycolysis

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GlycolysisGlycolysis Key points about the Shuttle System:

Malate-Asparate shuttle is the primary system By default Glycerol shuttle is secondary

Two enzymes are involved in this shuttle:

1.cytosolic version of the enzyme glycerol-3-phosphate

dehydrogenase (glycerol-3-PDH) which has as onesubstrate, NADH.

2.mitochondrial form of the enzyme which has as one of

its' substrates, FAD+. Since the electrons from

mitochondrial FADH2 feed into the oxidative

phosphorylation pathway at coenzyme Q (as opposed to

NADH-ubiquinone oxidoreductase [complex I]) only 2

moles of ATP will be generated from glycolysis. G3PDH

is glyceraldehyde-3-phoshate dehydrogenase.

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GlycolysisGlycolysis

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Malate -Asp Shuttle

The electrons are "carried" into the mitochondria inthe form of malate. Cytoplasmic malate

dehydrogenase (MDH) reduces oxaloacetate (OAA)to malate while oxidizing NADH to NAD+

Cytoplasmic malate dehydrogenase (MDH) reducesoxaloacetate (OAA) to malate while oxidizing NADHto NAD+.

Malate then enters the mitochondria where thereverse reaction is carried out by mitochondrial MDH

mitochondrial OAA goes to the cytoplasm to maintainthis cycle ; must be transaminated to aspartate (Asp)with the amino group being donated by glutamate(Glu). The Asp then leaves the mitochodria andenters the cytoplasm. The deamination of glutamategenerates a-ketoglutarate (a-KG) which leaves themitochondria for the cytoplasm.

When the energy level of the cell rises, the rate ofmitochondrial oxidation of NADH to NAD+ declines

and therefore, the shuttle slows.

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The synthesis ofF2,6BP is catalyzed by the bifunctionalenzymephosphofructokinase-2/fructose-2,6-bisphosphatase (PFK-2/F-2,6-BPase).

In the nonphosphorylated form the enzyme is known asPFK-2 and serves to catalyze the synthesis of F2,6BP byphosphorylating fructose 6-phosphate.

The result is that the activity of PFK-1 is greatlystimulated and the activity of F-1,6-BPase is greatly

inhibited. More glycolysis! When the bifunctional enzyme is phosphorylated it nolonger exhibits kinase activity, but a new active sitehydrolyzes F2,6BP to F6P and inorganic phosphate.

This enzyme is regulated by ProteinKinase A, which is acyclic AMP dependent enzyme. cAMP is generated

depending on the hormonal changes in the body. Eg.With Glucagon, high cAMP thus PKA is active thus lessglycolysis.

In addition to these Pyruvate Kinase is activated byF1,6BP and inhibited by ATP.

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genesisgenesis

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Substrates forGluconeogenesis: Lactate, pyruvate, glycerol, propionny-CoA and certain Amino

Acids but never FAT!!!

The Cori cycle involves the utilization of lactate, produced by glycolysis in non-hepatic tissues, (such as muscle and erythrocytes) as a carbon source for hepatic

gluconeogenesis. In this way the liver can convert the anaerobic byproduct of

glycolysis, lactate, back into more glucose for reuse by non-hepatic tissues. Note

that the gluconeogenic leg of the cycle (on its own) is a net consumer of energy,

costing the body 4 moles of ATP more than are produced during glycolysis.

Therefore, the cycle cannot be sustained indefinitely.

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The glucose-alanine cycle is used primarily as a mechanism for skeletal muscle to

eliminate nitrogen while replenishing its energy supply.Glucose oxidation produces

pyruvate which can undergo transamination to alanine. This reaction is catalyzed by

glutam

ate-pyruvate transam

inase, GPT (also called alanine tran

sam

inase, ALT inFigure).

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Regulation ofGluconeogenesis

See regulation ofGlycolysis via F2,6 P Do not forget Hormonal regulations: Insulin and

Glucagon

Other things to keep in mind

Pyruvate carboxylase is present in mitochondria, requires Biotin as

a cofactor to convert Pyruvate OAA

MDH present in mitochondria,OAA to malate, then MDH present

in cytosol converts malate back to OAA

OAA is then converted to PEP, as shown in the previous slide.

Pyruvate Carboxylase: inhibited by ADP and activated Acetyl CoA

PEP Carboxykinase in the cytosol is inhibited by ADP

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TCA /Citric Acid/KREBS CycleTCA /Citric Acid/KREBS Cycle The cycle is located in the mitochondria

All cells have a mitochondria except RBCs

This is the Final common pathway of oxidativemetabolism

Acetyl coenzyme A condenses with OAA to begin thecycle. Catabolism of CHO, Fats and Proteins provide theacetyl CoA

The bulk of ATP used by many cells to maintainhomeostasis is produced by the oxidation of pyruvate inthe TCA cycle

During this oxidation process, reduced NADH andreduced FADH2 are generated. The NADH and FADH2

are principally used to drive the processes ofoxidativephosphorylation, which are responsible for convertingthe reducing potential of NADH and FADH2 to the highenergy phosphate in ATP

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The PDH complex requiresThe PDH complex requires 5 different coenzymes:5 different coenzymes: CoACoA,, NAD+NAD+,, FAD+FAD+,, lipoicacidlipoicacid andand thiaminethiamine

pyrophosphate (TPP)pyrophosphate (TPP) . Three of the coenzymes of the complex are tightly bound to enzymes of the. Three of the coenzymes of the complex are tightly bound to enzymes of the

complex (TPP, lipoic acid and FAD+) and two are employed as carriers of the products of PDH complexcomplex (TPP, lipoic acid and FAD+) and two are employed as carriers of the products of PDH complex

activity (CoA and NAD+).activity (CoA and NAD+). ppyruvate + CoA + NAD+yruvate + CoA + NAD+ CO2 + acetylCO2 + acetyl--CoA + NADH + H+CoA + NADH + H+

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The TCA cycle showing enzymes, substrates and products. The abbreviated enzymes are: IDH =The TCA cycle showing enzymes, substrates and products. The abbreviated enzymes are: IDH = isocitrateisocitrate

dehydrogenasedehydrogenase and aand a--KGDH =KGDH = aa--ketoglutarate dehydrogenaseketoglutarate dehydrogenase. The GTP generated during the. The GTP generated during thesuccinatesuccinate

thiokinasethiokinase (succinyl(succinyl--CoA synthetase) reaction is equivalent to a mole of ATP by virtue of the presence ofCoA synthetase) reaction is equivalent to a mole of ATP by virtue of the presence of

nucleoside diphosphokinasenucleoside diphosphokinase. The 3 moles of NADH and 1 mole of FADH2 generated during each round of. The 3 moles of NADH and 1 mole of FADH2 generated during each round of

the cycle feed into thethe cycle feed into the oxidative phosphorylationoxidative phosphorylationpathway. Each mole of NADH leads to 3 moles of ATP andpathway. Each mole of NADH leads to 3 moles of ATP and

each mole of FADH2 leads to 2 moles of ATP.each mole of FADH2 leads to 2 moles of ATP.

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Overall Stoichiometry of TCAOverall Stoichiometry of TCA acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O ----> 2CO2 + 3NADH + FADH2 + GTP + 2H+ + HSCoA

The GTP generated by Succinyl CoA SYNTHETASE IS VIA SUBSTRATE LEVEL PHOSPORYLATION.

Regulation of TCA: Regulation ofthe TCA cycle like that ofglycolysis, occursat both thelevel ofentry ofsubstrates into the cycle as wellasat the key reactions ofthe cycle. Fuel entersthe TCA cycle primarilyasacetyl-CoA. The generation ofacetyl-CoA fromcarbohydrates isamajorcontrol point ofthe cycle. This is the reaction catalyzed by the PDH complex

PDH complex is inhibited byacetyl-CoA, ATP, and NADH

PDH activated by non-acetylated CoA (CoASH) and NAD+.

Thepyruvate dehydrogenase activities of the PDH complex are regulated bytheir state of phosphorylation. This modification is carried out by a specifickinase (PDH kinase) and the phosphates are removed by a specific phosphatase(PDH phosphatase).

The phosphorylation of PDH inhibits its activity which leads to decreasedoxidation of pyruvate.

PDH kinase is activated byN

ADH and acetyl-CoA and inhibited by pyruvate,ADP, CoASH, Ca2+ and Mg2+. ThePDH phosphatase, in contrast, is activatedby Mg2+ and Ca2+

Citrate Synthase: inhibited by ATP and citrate

Isocitrate Dehydrogenase: Isocitrate, AMP, ADP activates, ATP and NADH inhibits

A-ketoglutarate dehydrogenase: succinoyl CoA and NADH inhibits

CindyIsKinkySoSheFornicatesMoreOften

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ELECTRON TRANSPORT ANDELECTRON TRANSPORT AND

OXIDATIVE PHOSPHORYLATIONOXIDATIVE PHOSPHORYLATION

Each turn of TCA cycle generates 3NADH and 1 FADH2

Electron transport and oxophos occurs in themitochondria

NADH and FADH2 ultimately pass electrons to O2 andproduce H2O. NADH + (1/2)O2 + H+ -->NAD+ + H2O ~ -52.6kcal/mol

ADP + PATP ~ +7.3kcal/mol

Energy from NADH can be used to drive synthesis of ATPseveral times.

Important again to remember this is an oxidation-reduction reactionthus our friend Nerst is back:

DeltaG' = -nFDE'

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Electron Transport is coupled to OxidativeElectron Transport is coupled to Oxidative

PhosphorylationPhosphorylation The idea of coupling is explained by Mitchells

CHEMIOSMOTIC HYPOTHESIS

Basically coupling electron flow through the ETC to ATP synthesis

The Respiratory complexes are proton pumps. As electrons pass throughcomplexes I, III, and IV, hydrogen ions are pumped across the innermitochondrial membrane into the intermembrane space.

The proton concentration in the intermembrane space increases relative to themitochondrial matrix

This generates aproton-motive force as a result of 2 factors: 1) Difference in pHand 2) Difference in electrical potential, delta si, between intermembrane spaceand the mitochondrial matrix.

ATP synthetase complex (complex V): Hydrogen ions pass back into the matrixthrough V, this drives ATP synthesis.

NADH 3ATP

FADH2 2 ATP: note bypass of Complex 1

ATP synthesized in the matrix is transported out of the matrix via an ATP/ADPtranslocase (an antiport) also coupled to proton motive force.

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Inhibitors of Oxidative PhosphorylationInhibitors of Oxidative Phosphorylation

Rotenone: e- transport inhibitor Complex I

Amytal: e- transport inhibitor Complex I

Antimycin: A e- transport inhibitor Complex III

Cyanide: e- transport inhibitor Complex IV

Carbon Monoxide: e- transport inhibitor Complex IV Azide e- transport inhibitor Complex IV

2,4,-dinitrophenol: Uncoupling agent transmembrane H+carrier

Pentachlorophenol: Uncoupling agent transmembrane H+carrier

Oligomycin: Inhibits ATP synthase

Thermogenin: also an uncoupler, component of brown fat

Malonate inhibits Complex II

There are others in your handout take a look at them.

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SOME MORE STUFFSOME MORE STUFF

TCA cycle is regulated by the ratio of ADP, Pi/ ATP Under resting conditions, with a high cell energy charge, the

demand for new synthesis of ATP is limited and, although theProton Motive Force is high, flow of protons back into themitochondria through ATP synthetase is minimal. When energydemands are increased, such as during vigorous muscle activity,cytosolic ADP rises and is exchanged with intramitochondrialATP via the transmembrane adenine nucleotide carrierADP/ATPtranslocase. Increased intramitochondrialconcentrations ofADP cause the Proton Motive Force tobecome discharged as protons pour through ATPsynthetase, regenerating the ATP pool.

The rate of electron transport is dependent on the PMF

ANY BLOCKADE AT ANY POINT IN THE ELECTRONTRANSPORT CHAIN STOPS ATP SYNTHESIS!!!!!!!!!

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SAMPLE QUESTIONSSAMPLE QUESTIONS

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Choose the INCORRECT statement concerning the ATP-ADP cycle and the study of bioenergetics in the human body:

a. One half of the ATP-ADP cycle involves the coupling the

energy derived from the hydrolysis of the high energyphosphate bonds of ATP to endergonic reactions so that theywill occur spontaneously.

b. The work that requires energy derived from ATP hydrolysisincludes the transport of electrons down the electron

transport chain. c. One half of the ATP-ADP cycle involves the generation ofATP that starts with the formation of reduced coenzymes likeNADH and FADH2and the ultimate transfer of their electronsto oxygen

d. An important part of oxidative phosphorylation and ATPbiosynthesis is the generation of an electrochemical gradientacross the inner membrane of the mitochondria.

Many catabolic reactions, like the TCA cycle and fatty acidoxidation, provide the reduced coenzymes for the start ofoxidative phosphorylation and ATP biosynthesis

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Since electron transport and oxidative phosphorylationare tightly coupled, which one of the followingmechanisms BEST explains how ADP regulates the rateof electron transport during oxidative phosphorylation?

a. AMP concentrations are increased as ADPconcentrations fall

b. Low [ADP] accelerates the Krebs (TCA) cyclereaction rates, thereby providing more NADH to activateelectron transport

c. The transmembrane proton gradient is dissipatedwith low [ADP]

d. The ATP/ADP antiport system is not functional whenmitochondrial [ADP] is low

e. Proton translocation across the inner mitochondrialmembrane is decreased when ATP-synthase lacksbound ADP and Pi, secondarily retarding electrontransport

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You isolate mitochondria from a group of patientsthat present with lactic acidosis and muscleweakness, and show that they are unable to: (1)

oxidize reduced coenzyme Q, (2) translocate protonsacross their mitochondrial membranes to theintennembrane space against a concentrationgradient with succinate added as the substrate, and(3) reduce cytochrome c. The biochemical defect inthese patients most likely resides in their ... ?

A. Complex I (NADH dehydrogenase)

B. Complex II (succinate-Q reductase) C. Complex III (cytochrome b-c1)

D. Complex IV (cytochrome oxidase)

E. Complex V (F1F0 ATPase)

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Which of the following orderings #1 - #5 of the various componentsof the electron transport chain and oxidative phosphorylation willeffectively allow the development of an electrochemical potentialsufficient to drive the generation of high energy phosphate bonds

between ADP and Pi?

1. FMN, NADH dehydrogenase, ubiquinone, cytochrome c, cytochromeoxidase, F1F0-ATPase

2. Complex I, Complex III, ubiquinone, cytochrome a1-a3, cytochrome c,Complex IV, Complex V

3. FAD(2H)/succinate dehydrogenase, Coenzyme Q, cytochrome b-cl,cytochrome c, cytochrome a1-a3, F1F0-ATPase

4. NADH dehydrogenase, CoQ, cytochrome b-cl, cytochrome c,cytochrome oxidase, ATP synthase

5, NADH dehydrogenase, CoQ, cytochrome c, cytochrome oxidase,cytochrome b-cl, F1F0-ATPase

a. Both #1 and #2

b. Both #3 and #4

c. Only #4

d. Only #3

e. None of the above

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As a skilled cell biologist and biochemist, you cleverlydevise a method for experimentally separating the F1portion of ATP synthase from the membrane-bound

Fo fragment in intact mitochondria. Which of thefollowing metabolic effects do you observe?

a. Electron transport and oxygen consumption areinhibited

b. Electron transport and phosphorylation of ADPremain tightly coupled

c. The inner mitochondrial membrane remainsimpermeable to protons

d. Protons pass through the membrane-bound Fofragment, but they do not sustain any ATP formation

e. The F1 fragment forms ATP at an accelerated rateuntil ADP is depleted or the proton gradient is

dissipated

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Which of the following groups of enzymatic reactions,enzymes and substrates comprise importantanaplerotic pathways for 4-carbon intermediates

critical to the citric acid (TCA) cycle in the liver, muscleand nervous tissues?

a. conversion of pyruvate to acetyl CoA via pyruvatedehydrogenase and glutamate to a-ketoglutarate via

transaminases b. conversion of cc-ketoglutarate to glutamate andGABA

c. production of ketone bodies (acetoacetate and P-

hydroxybutyrate) d. conversion of pyruvate to oxaloacetate via

pyruvate carboxylase, biotin, bicarbonate ion, andATP

e. both (A) and (D)

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Regulation of tricarboxylic acid cycle

activity in vivo may involve the

concentration of all of the followingEXCEPT:

acetyl CoA

ADP.

ATP.

CoA.

oxygen.

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NAD+ can be regenerated in the cytoplasm if

NADH reacts with any of the following

EXCEPT:

pyruvate.

dihydroxyacetone phosphate.

oxaloacetate.

the flavin bound to NADH dehydrogenase.

phosphoglycerate kinase.

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Glucokinase:

has a Km considerably greater than thenormal blood glucose concentration..

is found in muscle.

is inhibited by glucose 6-phosphate. is also known as the GLUT-2 protein.

has glucose 6-phosphatase activity as well

as kinase activity.

I th C i l

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In the Cori cycle:

only tissues with aerobic metabolism (i.e.,mitochondria and O2) are involved.

a three-carbon compound arising form glycolysisis converted to glucose at the expense of energyfrom fatty acid oxidation.

glucose is converted pyruvate in anaerobic

tissues, and this pyruvate returns to the liver,where it is converted to glucose.

the same amount of ATP is used in the liver tosynthesize glucose as is released duringglycolysis, leading to no net effect on whole-body energy balance.

nitrogen from alanine must be converted to urea,increasing the amount of energy required todrive the process.

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A 7yr old female presents with anxiety,

dizziness, sweating and nausea following

brief periods of exercise. The symptomsare relieved by eating and do not occur if

the patient is frequently fed small meals.

Blood analysis indicates she is

hypoglycemic following brief period offasting, alanine fails to increase blood

sugar, fructose or glycerol administration

restores glucose to normal? What Pathway is affected, which enzyme

could it be? How would you confirm your

speculation?

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After the BIOCHEM exam you and your friendsdecided to only drink liquid-fire (Bacardi 151)for the rest of the evening. The next morningyou manage to wakeup with terrible hangover.

Which of these molecules is most responsiblefor your hangover?

Lactic Acid

Pyruvate

Acetate Acetyladehyde

Ethanol

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ADH alcohol dehydrogenase

AcDH acetyladehyde dehydrogenase

Acetaldehyde forms adducts with Proteins, nucleic acids, and othercompounds results in hangover.

NADH/NAD+ imbalance causes Liver to over work. Diversion of gluconeogenesis by Lactic Acid dehydrogenase decreasesability of Liver to deliver glucose to the blood.

In addition, there is increased synthesis of FAT. Acetate + CoA gives youacetyl-CoA which is a precursor for Fatty acid sythesis. You already haveenough NADH to go to work. So let the FATTYLIVER BEGIN!HepatoMEGALLY! Lets go!

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CLINICAL CORRELATIONSCLINICAL CORRELATIONS Riboflavin Deficiency

FMN and FAD are both synthesized from riboflavin, which contains the electron-

accepting ringstructure ofFAD Severe Riboflavin deficiencydecreases the ability ofmitochondria to generate ATPvia oxidative phosphorylation

In general, impairment ofComplex I (NADH Dehydrogenase) inducesformation ofmitochondria with structuralabnormalities.

Iron Deficiency Anemia Characterized bydecreasedlevels ofHb and other heme containing proteins in

blood.

Iron-containingcytochromesand Fe-S centers ofETCare decreasedas well. Fatigue partly due to impaired ETCfor ATP generation

ETC inhibitorsat specificsites Rotenone andAmytalblock Complex I

Antimycin blockscytochrome b1 in Complex III

Cyanide blockscytochrome a/a3 in Complex IV. Prevents reduction ofe- fromreduced cytochrome c.

CObinds to reduced iron ofcytochrome oxidase

Cyanide Poisoning CN- causesa rapid and extensive inhibition ofETCat the cytochrome oxidase step.

PreventsO2 fromservingas the final e- acceptor.

Mitochondrial respiration and energy production cease, resulting in cell death

Occursfrom tissue asphyxiation, most notably in the Nervous System

Treatment: nitritesadministered to convert oxyHb to MetheHb, which can then

compete with cytochrome a,a3 for the CN-, formingacomplex.

Oxidative Phosphorylation II the uncoupling of ETC and Ox Phos

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Oxidative Phosphorylation II the uncoupling of ETC and Ox-Phos

Uncoupling of ETC with Ox-Phos

Proton gradient from ETC coupled to ATP production from OxidativePhosphorylation. If uncoupled and proton gradient dissipated, ATPand ADP concentrations lose their ability to regulate the rate of e-

transport. Uncouplers: proton ionophores, which rapidly transport H+ from

cytosolic to matrix side of inner mitochondiral membrane

DNP picks up H+ on cyto side, drops H+ on matrix side

Oligomycin: inhibits F1F0-ATPaseATPsynthesisstops.

Respiration and transport are blocked

Addition of an uncoupler (DNP) induces initiation ofO2consumptionETC continues but w/o ATP synthesis since the pathwaysare uncoupled.

Brown Adipose Tissue and Thermogenesis

Large deposits of brown fat around vital organs (in humaninfants)specialized for non-shivering thermogenesis.

Cold or excessive food intake stimulates NE release Then Thermogenin, proton conductance uncoupler, is activated,

pumping H+ back into mitochondriadissipating the gradient.

ETC is induced, increasing rate of NADH and FADH2 oxidation,which generates more heat = biological heating pad

Hyperthyroidism Graves Disease

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Hyperthyroidism Grave s Disease

Thyroid hormone influences bioenergetics via actions on mitochondrialox phos.

In Hyperthyroidism, energy derived from ox. Phos is significantly lessthan normal.

Thryoid causes uncoupling ofOx Phos. Results in increased heat production patients complain of feeling hot

and sweaty.

Salicylate (aspirin) poisoning

At high concentrations, salicylate can partially uncouple mitochondrialOx Phos.

DecreasedATP [ ] and increasedcytosolic AMP induce glycolysis Results in increasedblood pyruvate and lactate and metabolic acidosis

and fever

Myoclonic Epileptic Ragged Red Fiber Disease (MERRF)

Debilitating, progressive spontaneous muscle jerking

Mitochondrial myopathy with enlarged, abnormal mitochondria

Neurosensory hearing loss, dementia, hypoventilation, mildcardiomyopathy

Maternal inheritance (sex linked)

Impaired energy metabolism.lactic acidosis

Pentose Phosphate Pathway

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Pentose Phosphate Pathway

Hemolysiscaused by Reactive Oxygen Species(ROS) G6PD deficiency in pentose phosphate pathway

Causes increased production of radicals from GSH, since cant producesufficient NADPH to re-reduce glutathione.result in hemolysis

Heinz Bodies in RBCs Due to G6PD deficiency

RBCs need the enzyme to re-reduce glutathione with NADPH to protectagainst oxidative stress

ROS peroxidation of membrane lipids lyses the RBC membrane

G6PD Mediterranean disease most severe G6PD deficiency

Lecture 21 Monosaccharidesand interconversion ofsugars ClassicalGalactosemia

Deficiency ofGalactosyl-1-P uridylyltransferase

Accumulation ofG-1-P in tissues and inhibition of glycogen metabolism,which require UDP-sugars

Higher level of galactose in blood and urine

More serious form Non-ClassicalGalactosemia

Galactokinase deficiency

Unable to convert galactose to galactose-1-P

Glycogen Synthesis

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Glycogen Synthesis

Glucose Toxicity Dysfunction of glycogen synthase

Due to hyperglycemiaproduces insulin resistance

Due to production of hexosamines that inhibit hexokinase, protein phosphatase 1, andglycogen synthase.

Lecture 23 Glycogen Degradation

Von Gierkes Disease Defective G-6-Phosphatase enzyme

Increased amount of glycogen, normal structure

Affects liver and kidney

Massive enlargement of the liver. Severe hypoglycemia, ketosis, hyperuricemia,hyperlipemia.

Lecture 24 Glucose/Glycogen Regulation

Type I Insulin-dependent diabetesmellitus Hyperglycemic

Continuous glucagon expression causes ketogenesis, lipolysis, and gluconeogenesis.

Hyperchylomicronemia occurs (liver TG syn and VLDL transport faster than adipose LPLbreakdown of TG)

Risk of ketoacidosis Type II Noninsulin-dependent Diabetes Mellitus Hyperglycemic

Peripheral tissues insulin resistant

Glucose accumulates in blood due to poor uptake by peripheral tissues, particularlymuscles

Hypertriacylglycerolemia, which results from increase of VLDL withouthyperchylomicronemia. New FA and VLDL synthesized in liver instead of increaseddelivery of fatty acids from adipose tissue.

en ose osp a een ose osp a e

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PathwayPathway

What is the PPP and why is it important?


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PathwayPathway

What is the PPP and why is it important?

The pentose phosphate pathway is primarily an anabolic pathway that utilizes the

6 carbonsofglucose to generate 5 carbon sugars and reducing equivalents

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Pentose Phosphate PathwayPentose Phosphate Pathway

To generate reducing equivalents, in the form ofNADPH, for reductive biosynthesis reactions within cells

To provide the cell with ribose-5-phosphate (R5P) for the

synthesis of the nucleotides and nucleic acids

Although not a significant function of the PPP, it canoperate to metabolize dietary pentose sugars derivedfrom the digestion of nucleic acids as well as torearrange the carbon skeletons of dietary carbohydratesinto glycolytic/gluconeogenic intermediates

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Pentose Phosphate PathwayPentose Phosphate Pathway

The reactions of fatty acid biosynthesis and steroid biosynthesisutilize large amounts of NADPH. As a consequence, cells of the liver,adipose tissue, adrenalcortex, testisand lactatingmammarygland have high levels ofthe PPP enzymes.

Erythrocytes utilize the reactions of the PPP to generate largeamounts of NADPH used in the reduction of glutathione

The conversion of ribonucleotides to deoxyribonucleotides (throughthe action ofribonucleotide reductase) requires NADPH as the

electron source, therefore, any rapidly proliferatingcell needslarge quantities ofNADPH

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PathwayPathway

The reactions of the PPP operate exclusively in the cytoplasm. From this

perspective it is understandable that fatty acid synthesis (as opposed to

oxidation) takes place in the cytoplasm

The oxidation steps, utilizing glucose-6-phosphate (G6P) as the substrate,occur at the beginning of the pathway and are the reactions that generate

NADPH

Reactionscatalyzed byglucose-6-phosphate dehydrogenase and 6-

phosphogluconate dehydrogenase generate one mole ofNADPH each for

everymole ofglucose-6-phosphate (G6P) that enters the PPP

Oxidative Pathway


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PathwayPathwayNon-oxidative reactions are to convert dietary 5 carbon sugars into both 6 (fructose-6-

phosphate) and 3 (glyceraldehyde-3-phosphate) carbon sugars which can then be

utilized by the pathways ofglycolysis

The primary enzymes involved in the non-oxidative steps of the PPP are transaldolase

and transk

eto

lase

Transketolase functions to transfer 2 carbon groups from substrates of the PPP,thus rearranging the carbon atoms that enter this pathway. Like other enzymes that

transfer 2 carbon groups, transketolase requires thiamine pyrophosphate (TPP) as a co-

factor in the transfer reaction

Transaldolase transfers 3 carbon groups and thus is also involved in arearrangement of the carbon skeletons of the substrates of the PPP. The transaldolase

reaction involves Schiff base formation between the substrate and a lysine residue in the

enzyme

Non-oxidative Pathway

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PathwayPathwayWhats the point?


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PathwayPathwayWhats the point?

R5Pproduction

Oxidation ofG6P, a 6 carbon sugar, into a 5 carbon sugar

Generation ofNADPH

3 carbon sugar generated is glyceraldehyde-3-phsphate whichcan be shunted to glycolysis and oxidized to pyruvate OR it can

be utilized by the gluconeogenic enzymes to generate more 6

carbon sugars (fructose-6-phosphate or glucose-6-phosphate)


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PathwayPathwayRBCsand the PPPPredominant pathways of carbohydrate metabolism in the red blood cell (RBC) are

glycolysis, the PPP and 2,3-bisphosphogylcerate (2,3-BPG)

Glycolysis provides ATP for membrane ion pumps and NADH for re-oxidation of

methemoglobin

The PPP supplies the RBC with NADPH to maintain the reduced state of

glutathione (Glutathione can reduce disulfides nonenzymatically)

Oxidative stress generates peroxides that in turn can be reduced byglutathione to

generate water

Inability to maintain reduced glutathione in RBCs leads to increased accumulation of

peroxides, predominantly H2O2, that in turn results in a weakening of the cell wall and

concomitant hemolysis

Glutathione removes peroxides via the action ofglutathione peroxidase. The PPP in

erythrocytes is essentially the only pathwayfor these cells to produce NADPH

Glycogen MetabolismGlycogen Metabolism

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Glycogen MetabolismGlycogen Metabolism

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Glycogen is a polymer ofglucose residues linked by

E(14) glycosidic bonds, mainly

E(16) glycosidic bonds, at branch points

Glycogen chains & branches are longer than shownGlucose is stored as glycogen predominantly in liverand muscle cells.

H O

OH

H

OHH

OH

CH2OH

HO H

H

OHH

OH

CH2OH

H

O

HH H O

OH

OHH

OH

CH2

HH H O

H

OHH

OH

CH2OH

H

OH

HH O

OH

OHH

OH

CH2OH

H

O

H

O

1 4

6

H O

H

OHH

OH

CH2OH

HH H O

H

OHH

OH

CH2OH

HH

O

1

OH

3

4

5

2

glycogen

C O

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Glycogen Phosphorylase catalyzes phosphorolytic

cleavage of the E(14) glycosidic linkages ofglycogen, releasing glucose-1-phosphate asreaction product.

glycogen(n residues) + Pi

glycogen (n1 residues) + glucose-1-phosphate

glucose-1-phosphate

H O

OH

H

OHH

OH

CH2OH

H

OPO32

HGlycogen

catabolism(breakdown):

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Commonly used terminology:

"a" is the form of the enzyme that tends to be active, andindependent of allosteric regulators (in the case ofGlycogen

Phosphorylase, when phosphorylated).

"b" is the form of the enzyme that is dependent on local allosteric

controls (in the case ofGlycogen Phosphorylase when

dephosphorylated).

Glycogen catabolism

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Most people dont know

The relative activity of the un-modified phosphorylase enzyme(phosphorylase-b) is sufficient to generate enough glucose-1-phosphate for entry into glycolysis for the production ofsufficient ATP to maintain the normal restingactivity of thecell; This is true in both liver and muscle cells

Glycogen catabolism

Gl Ph h l i l i bj

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Glycogen Phosphorylase in muscle is subject toallosteric regulation by AMP, ATP, and glucose-6-phosphate. A separate isozyme of Phosphorylase

expressed in liver is less sensitive to these allostericcontrols.

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, inhibitPhosphorylase, promoting the tense conformation.

Thus glycogen breakdown is inhibited when ATPand glucose-6-phosphate are plentiful.

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Regulation bycovalent modification (phosphorylation):

The hormones glucagon and epinephrine activate G-protein coupled receptors to triggercAMP cascades.

Both hormones are produced in response to lowblood sugar.

Glucagon, which is synthesized by

E-cells of thepancreas, activates cAMP formation in liver.

Epinephrine activates cAMP formation in muscle.

Glycogen catabolism

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In response to lowered blood glucose the a cells of the pancreas secrete glucagon

which binds to cell surface receptors on liver and several other cells; Liver cells are

the primary target for the action of this peptide hormone

Activation of the enzyme adenylate cyclase which leads to a large increase in the

formation of cAMP

cAMP binds to an enzyme called cAMP-dependent protein kinase, PKA. This

leads to PKA-mediated phosphorylation ofphosphorylase kinase Phosphorylasekinase activates the enzyme which in turn phosphorylates the b form of

phosphorylase

Phosphorylation ofphosphorylase-b greatly enhances its activity towards glycogen

breakdown (phosphorylase-a)

The net result is an extremely large induction of glycogen breakdown in response

to glucagon binding to cell surface receptors

Glycogen catabolism

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Hormone (epinephrine or glucagon)

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Signal

cascade by

which

Glycogen

Phosphorylase

is activated.

via G Protein (GE-GTP)

Adenylate cyclase Adenylate cyclase

(inactive) (active)

catalysis

ATP cyclic AMP + PPi

Activation Phosphodiesterase

AMP

Protein kinase A Protein kinase A(inactive) (active)

ATP

ADP

Phosphorylase kinase Phosphorylase kinase (P)(b-inactive) (a-active)

Phosphatase ATPPi ADP

Phosphorylase Phosphorylase (P)

(b-allosteric) (a-active)

Phosphatase

Pi

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The cAMP cascade results in phosphorylation of a serinehydroxyl ofGlycogen Phosphorylase, which promotes transition tothe active (relaxed) state.

The phosphorylated enzyme is lesssensitive to allostericinhibitors.

Thus, even ifcellular ATP & glucose-6-phosphate are high,

Phosphorylase will be active.

The glucose-1-phosphate produced from glycogen in liver may beconverted to free glucose for release to the blood.

With this hormone-activated regulation, the needs of the organismtake precedence over needs of the cell.

Glycogen catabolism

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This identical cascade of events occurs in skeletalmuscle cells

However, in these cells the induction of the cascade is the result of

epinephrine binding to receptors on the surface of muscle cells(Ca2+ ion-mediated pathway tophosphorylase kinase activation is through activation of

a-adrenergic receptors by epinephrine)

Epinephrine is released from the adrenal glands in response to neural

signals indicating an immediate need for enhanced glucose utilizationin muscle, the so called fight orflight response

Muscle cellslack glucagon receptors. The presence of glucagonreceptors on muscle cells would be futile anyway since the role of

glucagon release is to increase blood glucose concentrations andmuscle glycogen stores cannot contribute to blood glucoselevelswhy?

Glycogen catabolism

Glycogen catabolism

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Regulation ofphosphorylase kinase activity is also affected by two distinct

mechanisms involving Ca2+ ions

The ability of Ca2+ ions to regulatephos

pho

rylasekina

se is through theubiquitous protein, calmodulin

Calmodulin is a calcium binding protein; binding induces a conformational

change in calmodulin which in turn enhances the catalytic activity of the

phosphorylase kinase towards its substrate,phosphorylase-b.

This activity is crucial to the enhancement of glycogenolysis in muscle cells

where muscle contraction is induced via acetylcholine stimulation at theneuromuscular junction

The effect ofacetylcholine release from nerve terminals at a neuromuscular

junction is to depolarize the muscle cell leading to increased release of

sarcoplasmic reticulum stored Ca2+, thereby activatingphosphorylase

kinase

Thus, not only does the increased intracellular calcium increase the rate ofmuscle contraction it increases glycogenolysis which provides the muscle cell

with the increased ATP it also needs for contraction

Glycogen catabolism

Ph h l Ki i i

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Phosphorylase Kinase in muscle includes calmodulin as its H subunit.Phosphorylase Kinase is partly activated by binding ofCa++ to this subunit

Phosphorylation of the enzyme, via a cAMP cascade induced byepinephrine, results in furtheractivation

These regulatory processes ensure release of phosphorylated glucose fromglycogen, for entry into Glycolysis to provide ATP needed for musclecontraction.

Phosphorylase Kinase inactive

Phosphorylase Kinase-Ca++ partly active

P-Phosphorylase Kinase-Ca++

fully active

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Pyridoxal phosphate (PLP), a

derivative of vitamin B6, serves as

prosthetic group forGlycogenPhosphorylase.

pyridoxal phosphate (PLP)

NH

CO

P

OO

O

OH

CH3

CH O

H2

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Question: Why would an inhibitor ofGlycogenPhosphorylase be a suitable treatment for diabetes?

A class of drugsdeveloped for treatingthe hyperglycemia ofdiabetes (chloroindole-carboxamides), inhibitliver Phosphorylase

allosterically.

These inhibitorsbindat the dimer interface,stabilizing the inactive

(tense) conformation.

PLP

PLP

GlcNAc

GlcNAc

inhibitor

Human LiverGlycogen Phosphorylase PDB 1EM6

D b hi h 2 i d d t ti it

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Debranching enzyme has 2 independent active sites,consisting of residues in different segments of a singlepolypeptide chain:

The transferase of the debranching enzyme transfers 3glucose residues from a 4-residue limit branch to theend of another branch, diminishing the limit branch to asingle glucose residue

The E(16) glucosidase moiety of the debranchingenzyme then catalyzes hydrolysis of the E(16) linkage,yielding free glucose. This is a minor fraction ofglucose released from glycogen

The major product of glycogen breakdown is glucose-1-phosphate, from Phosphorylase activity.

Enzyme-Ser-OPO32 Enzyme-Ser-OPO3

2Enzyme-Ser-OH

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Phosphoglucomutase catalyzes this reversible

reaction

glucose-1-phosphate glucose-6-phosphate

H O

OH

H

OHH

OH

CH2OH

H

OPO32

H H O

OH

H

OHH

OH

CH2OPO32

H

OH

HH O

OH

H

OHH

OH

CH2OPO32

H

OPO32

H

Enzyme Ser OPO3 Enzyme Ser OPO3Enzyme Ser OH

Glycogen Glucose

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The product glucose-6-phosphate may enterGlycolysis or (in liver) bedephosphorylated for release to the blood

LiverGlucose-6-phosphatase catalyzes the following, essential to the

liver's role in maintaining blood glucose:glucose-6-phosphate + H2O glucose + Pi

Most other tissueslack this enzymewhy??

Hexokinase or Glucokinase

Glucose-6-PaseGlucose-1-P Glucose-6-P Glucose + Pi

GlycolysisPathway

Pyruvate

Glucose metabolism in liver.

O

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Uridine diphosphate glucose (UDP-glucose) is theimmediate precursor forglycogen synthesis

As glucose residues are added to glycogen, UDP-glucose isthe substrate and UDP is released as a reaction product.

OO

OHOH

HH

H

CH2

H

HN

NO

OP

O

O

P

O

O

HO

OH

H

OHH

OH

CH2OH

H

O

H

UDP-glucose

Glycogensynthesis

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OO

OHOH

HH

H

CH2

H

HN

N

O

O

OP

O

O

P

O

O

H O

OH

H

OHH

OH

CH2OH

H

O

H

OP

O

O

HO

OH

H

OHH

OH

CH2OH

H

O

H

OO

OHOH

HH

H

CH2

H

HN

N

O

O

OP

O

O

P

O

O

OPO

O

O

PPi

+

UDP-glucose

glucose-1-phosphate UTP

UDP-Glucose Pyrophosphorylase

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UDP-glucose is formed from glucose-1-phosphate:

glucose-1-phosphate + UTP UDP-glucose + PPi

PPi + H2O

2 PiOverall:

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

Spontaneous hydrolysis of the ~P bond in PPi (P~P) drives the overallreaction

Cleavage of PPi is the only energy cost for glycogen synthesis (one ~Pbond per glucose residue).

Glycogenin initiatesglycogen synthesis.

Glycogenin is an enzyme that catalyzes glycosylation of one of its own

tyrosine residues.

CH2OH6 tyrosine residue

UDP-glucose

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A glycosidic bond is formed between the anomeric C1 of theglucose moiety derived from UDP-glucose and the hydroxyloxygen of a tyrosine side-chain ofGlycogenin.

UDP is released as a product.

H O

OH

H

OHH

OH

CH2OH

H

O H

H

OHH

OH

CH2OH

H

O

HHC

CH

NH

CH2

O

O

H O

OH

H

OHH

OH

CH2OH

H

H

C

CH

NH

CH2

O

O1

5

4

3 2

6

H O

OH

H

OHH

OHH

H

O1

5

4

3 2

P O P O Uridine

O

O

O

O

C

CH

NH

C

H2

HO

O

of Glycogenin

O-linkedglucoseresidue

+ UDP

UDP-glucose

CH2OH6

O-linked

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Glycosylation at C4 of the O-linked glucose product yields an O-linked

disaccharide with E(1p4) glycosidic linkage. UDP-glucose is again theglucose donor

This is repeated until a short linear glucose polymer with E(1p4)glycosidic linkages is built up on Glycogenin

H O

OH

H

OHH

OH

CH2OH

H

O H

H

OHH

OH

CH2OH

H

O

HHC

CH

NH

CH2

O

O

H O

OH

H

OHH

OHH

HC

CH

NH

C

H2

O

O1

5

4

3 2

UDP-glucose

O-linkedglucoseresidue

E(1 4)linkage

+ UDP

+ UDP

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Glycogen Synthase catalyzes transfer of theglucose moiety of UDP-glucose to the hydroxyl atC4 of the terminal residue of a glycogen chain toform an E(1p 4) glycosidic linkage:

glycogen(n residues)

+ UDP-glucose

glycogen(n +1 residues) + UDP

A separate branching enzyme transfers a segment

from the end of a glycogen chain to the C6 hydroxyl ofa glucose residue of glycogen to yield a branch with an

E(1p6) linkage.

Glycogen Synthesis

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Both synthesis & breakdown of glycogen are spontaneous

If both pathways were active simultaneously in a cell, there would be a "futile

cycle" with cleavage ofone ~P bond percycle (in forming UDP-glucose)

To prevent such a futile cycle, Glycogen Synthase and Glycogen Phosphorylase

are reciprocally regulated, by allosteric effectors and by phosphorylation.

Glycogen Synthesis

UTP UDP + 2 Pi

glycogen(n) + glucose-1-P glycogen(n + 1)

GlycogenPhosphorylase Pi

Glycogen Glucose

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Glycogen Synthase is allosterically activated by glucose-6-P(opposite of effect on Phosphorylase)

Thus Glycogen Synthase is active when high blood glucose leads toelevated intracellularglucose-6-P

It is useful to a cell to store glucose as glycogen when the input toGlycolysis (glucose-6-P), and the main product ofGlycolysis (ATP), areadequate.


Glucose-6-Pase

Glucose-1-P Glucose-6-P Glucose + PiGlycolysisPathway

Pyruvate


Glycogen Glucose

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High cytosolic glucose-6-phosphate, which would result when bloodglucose is high, turns off the signal with regard to glycogen synthesis

The conformation ofGlycogen Synthase induced by the allosteric

activator glucose-6-phosphate is susceptible to dephosphorylation byProtein Phosphatase (PP1)


Glucose-6-Pase

Glucose-1-P Glucose-6-P Glucose + PiGlycolysisPathway

Pyruvate


The cAMP cascade induced in liver by glucagon or epinephrine has

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The cAMP cascade induced in liver by glucagon or epinephrine hasthe opposite effect on glycogen synthesis.

Glycogen Synthase is phosphorylated by Protein Kinase A as well

as by Phosphorylase Kinase.Phosphorylation ofGlycogen Synthase promotes the "b" (lessactive) conformation.

The cAMP cascade thus inhibitsglycogen synthesis.

Instead of being converted to glycogen, glucose-1-P in liver may beconverted to glucose-6-P, and dephosphorylated for release to theblood.

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I li d d i t hi h bl d l t i

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Insulin, produced in response to high blood glucose, triggers a

separate signal cascade that leads to activation ofPhosphoprotein 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

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Glycogen StorageDiseases are genetic

enzyme deficienciesassociated with excessiveglycogen accumulationwithin cells

Some enzymes whosedeficiency leads to glycogenaccumulation are part of theinter-connected pathwaysshown here

glycogen

glucose-1-P

Glucose-6-Phosphatase

glucose-6-P glucose + Pi

fructose-6-P

Phosphofructokinase

fructose-1,6-bisP

Glycolysis continued

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When an enzyme defect affects mainly glycogenstorage in liver, a common symptom is

hypoglycemia, relating to impaired mobilizationof glucose for release to the blood during fasting.

When the defect is in muscle tissue, weakness

& difficulty with exercise result from inability toincrease glucose entry into Glycolysis duringexercise.

Additional symptoms depend on the particular

enzyme that is deficient.

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Glycogen Storage DiseaseSymptoms, in addition to

glycogen accumulation

Type I, liver deficiency ofGlucose-6-phosphatase (von

Gierke's disease)

hypoglycemia (low blood

glucose) when fasting, liver

enlargement.

Type IV, deficiency of

branching enzyme in variousorgans, including liver

(Andersen's disease)

liver dysfunction and early

death.

Type V, muscle deficiency of

Glycogen Phosphorylase

(McArdle's disease)

muscle cramps with exercise.

Type VII, muscle deficiency ofPhosphofructokinase.

inability to exercise.

SS

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SummarySummary

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bio chem 2

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