Post on 06-Apr-2018
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Medical BiochemistryMedical Biochemistry
Review #2Review #2
ByByJason ElmerJason Elmer
jelmer1@uic.edujelmer1@uic.edu
Obi EkwennaObi Ekwenna
oekwen1@uic.eduoekwen1@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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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.
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + PiGlycolysisPathway
Pyruvate
Glucose metabolism in liver.
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)
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + PiGlycolysisPathway
Pyruvate
Glucose metabolism in liver.
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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