Download Medical Biochemistry Review #2 By

Medical Biochemistry
Review #2
By
Jason Elmer
[email protected]
Obi Ekwenna
[email protected]
YOUR EXAM
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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!
It is impossible to memorize
every possible bit of
biochemistry trivia. ‘They’
simply know way too much
about metabolism for a single
person to be able to
regurgitate it all.
• 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
Do prioritize:
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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 interaction
of specific regulatory molecules in a pathway?
• What biochemical techniques are used to study these pathways?
• What specific drugs or diseases associated with the pathway?
METABOLIC PATHWAYS
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Glycolysis
Gluconeogenesis
Citric Acid Cycle (Krebs Cycle)
Glycogen Metabolism
Hexose Interconversions
Electron Transport Chain
Oxidative Phosphorylation
Pentose-Phosphate Shunt
GLYCOLYSIS
• Oxidation of glucose is known as Glycolysis.
– Either AerobicPyruvate
– 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 fuel
mitochondrial ATP synthesis via oxidative phosphorylation.
Does not pass through mitochondrial membrane
2 ATP generated glycerol phosphate shuttle
3 ATP generated malate-aspartate shuttle
If used to transport the electrons from cytoplasm NADH into
the mitochondria.
Key Reactions
• Hexokinase
– Found in the cytosol of most tissues
– Low specificity: it’s a “hoe” for hexoses
– Low Km: high affinity for glucose
– Inhibited by Glucose-6-phosphate
Glucokinase: Found in the Liver and pancreatic b
cells
Also a ‘hexokinase’
High specificity for glucose
High Km
inhibited by fructose-6-phosphate
Regulation of Glycolysis
• Hexokinase, PFK-1 and PK all proceed with a
relatively large free energy decrease. These nonequilibrium reactions of glycolysis would be ideal
candidates for regulation of the flux through
glycolysis.
• Hexokinase is not key because of G6P is generated by
glycogenolysis
• PK reaction is reversed in Gluconeogenesis
• Therefore rate limiting step in glycolysis is the
reaction catalyzed by PFK-1.
• PFK-1 is a tetrameric enzyme that exist in two
conformational states termed R and T that are in
equilibrium.
• ATP is both a substrate and an allosteric inhibitor of
PFK-1. F6P is the other substrate for PFK-1 and it also binds
preferentially to the R state enzyme. ATP binds the T state.
• 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 regulator of both
glycolysis and gluconeogenesis is fructose 2,6bisphosphate, 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.
Glycolysis
Glycolysis
• 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 one
substrate, 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.
Glycolysis
• Malate -Asp Shuttle
– The electrons are "carried" into the mitochondria in
the form of malate. Cytoplasmic malate
dehydrogenase (MDH) reduces oxaloacetate (OAA)
to malate while oxidizing NADH to NAD+
– Cytoplasmic malate dehydrogenase (MDH) reduces
oxaloacetate (OAA) to malate while oxidizing NADH
to NAD+.
– Malate then enters the mitochondria where the
reverse reaction is carried out by mitochondrial MDH
– mitochondrial OAA goes to the cytoplasm to maintain
this cycle ; must be transaminated to aspartate (Asp)
with the amino group being donated by glutamate
(Glu). The Asp then leaves the mitochodria and
enters the cytoplasm. The deamination of glutamate
generates a-ketoglutarate (a-KG) which leaves the
mitochondria for the cytoplasm.
– When the energy level of the cell rises, the rate of
mitochondrial oxidation of NADH to NAD+ declines
and therefore, the shuttle slows.
• The synthesis of F2,6BP is catalyzed by the bifunctional
enzyme phosphofructokinase-2/fructose-2,6bisphosphatase (PFK-2/F-2,6-BPase).
• In the nonphosphorylated form the enzyme is known as
PFK-2 and serves to catalyze the synthesis of F2,6BP by
phosphorylating fructose 6-phosphate.
• The result is that the activity of PFK-1 is greatly
stimulated and the activity of F-1,6-BPase is greatly
inhibited. More glycolysis!
• When the bifunctional enzyme is phosphorylated it no
longer exhibits kinase activity, but a new active site
hydrolyzes F2,6BP to F6P and inorganic phosphate.
• This enzyme is regulated by ProteinKinase A, which is a
cyclic AMP dependent enzyme. cAMP is generated
depending on the hormonal changes in the body. Eg.
With Glucagon, high cAMP thus PKA is active thus less
glycolysis.
• In addition to these Pyruvate Kinase is activated by
F1,6BP and inhibited by ATP.
G
l
u
c
o
n
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o
genesis
Substrates for Gluconeogenesis: Lactate, pyruvate, glycerol, propionny-CoA and certain Amino
Acids but never FAT!!!
•The Cori cycle involves the utilization of lactate, produced by glycolysis in nonhepatic 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.
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
glutamate-pyruvate transaminase, GPT (also called alanine transaminase, ALT in
Figure).
• Regulation of Gluconeogenesis
• See regulation of Glycolysis 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
TCA /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 oxidative
metabolism
• Acetyl coenzyme A condenses with OAA to begin the
cycle. Catabolism of CHO, Fats and Proteins provide the
acetyl CoA
• The bulk of ATP used by many cells to maintain
homeostasis is produced by the oxidation of pyruvate in
the TCA cycle
• During this oxidation process, reduced NADH and
reduced FADH2 are generated. The NADH and FADH2
are principally used to drive the processes of oxidative
phosphorylation, which are responsible for converting
the reducing potential of NADH and FADH2 to the high
energy phosphate in ATP
The PDH complex requires 5 different coenzymes: CoA, NAD+, FAD+, lipoic acid and thiamine
pyrophosphate (TPP) . 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 complex
activity (CoA and NAD+).
pyruvate + CoA + NAD+  CO2 + acetyl-CoA + NADH + H+
The TCA cycle showing enzymes, substrates and products. The abbreviated enzymes are: IDH = isocitrate
dehydrogenase and a-KGDH = a-ketoglutarate dehydrogenase. The GTP generated during the succinate
thiokinase (succinyl-CoA synthetase) reaction is equivalent to a mole of ATP by virtue of the presence of
nucleoside diphosphokinase. The 3 moles of NADH and 1 mole of FADH2 generated during each round of
the cycle feed into the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP and
each mole of FADH2 leads to 2 moles of ATP.
Overall Stoichiometry of TCA
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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.
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Regulation of TCA: Regulation of the TCA cycle like that of glycolysis, occurs at both the
level of entry of substrates into the cycle as well as at the key reactions of the cycle. Fuel enters
the TCA cycle primarily as acetyl-CoA. The generation of acetyl-CoA from carbohydrates is a
major control point of the cycle. This is the reaction catalyzed by the PDH complex
– PDH complex is inhibited by acetyl-CoA, ATP, and NADH
– PDH activated by non-acetylated CoA (CoASH) and NAD+.
– The pyruvate dehydrogenase activities of the PDH complex are regulated by
their state of phosphorylation. This modification is carried out by a specific
kinase (PDH kinase) and the phosphates are removed by a specific phosphatase
(PDH phosphatase).
– The phosphorylation of PDH inhibits its activity which leads to decreased
oxidation of pyruvate.
– PDH kinase is activated by NADH and acetyl-CoA and inhibited by pyruvate,
ADP, CoASH, Ca2+ and Mg2+. The PDH phosphatase, in contrast, is activated
by 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
ELECTRON TRANSPORT AND
OXIDATIVE PHOSPHORYLATION
• Each turn of TCA cycle generates 3NADH and 1 FADH2
• Electron transport and oxophos occurs in the
mitochondria
• NADH and FADH2 ultimately pass electrons to O2 and
produce H2O.
– NADH + (1/2)O2 + H+ -->NAD+ + H2O ~
-52.6kcal/mol
– ADP + PATP ~ +7.3kcal/mol
– Energy from NADH can be used to drive synthesis of ATP
several times.
Important again to remember this is an oxidation-reduction reaction
thus our friend Nerst is back:
DeltaG' = -nFDE'
Electron Transport is coupled to Oxidative
Phosphorylation
• The idea of coupling is explained by Mitchell’s
CHEMIOSMOTIC HYPOTHESIS
– Basically coupling electron flow through the ETC to ATP synthesis
– The Respiratory complexes are proton pumps. As electrons pass through
complexes I, III, and IV, hydrogen ions are pumped across the inner
mitochondrial membrane into the intermembrane space.
– The proton concentration in the intermembrane space increases relative to the
mitochondrial matrix
– This generates a proton-motive force as a result of 2 factors: 1) Difference in pH
and 2) Difference in electrical potential, delta si, between intermembrane space
and the mitochondrial matrix.
– ATP synthetase complex (complex V): Hydrogen ions pass back into the matrix
through 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/ADP
translocase (an antiport) also coupled to proton motive force.
Inhibitors of Oxidative Phosphorylation
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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.
SOME 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 the
Proton Motive Force is high, flow of protons back into the
mitochondria through ATP synthetase is minimal. When energy
demands are increased, such as during vigorous muscle activity,
cytosolic ADP rises and is exchanged with intramitochondrial
ATP via the transmembrane adenine nucleotide carrier ADP/ATP
translocase. Increased intramitochondrial concentrations of
ADP cause the Proton Motive Force to become discharged
as protons pour through ATP synthetase, regenerating the
ATP pool.
– The rate of electron transport is dependent on the PMF
– ANY BLOCKADE AT ANY POINT IN THE ELECTRON
TRANSPORT CHAIN STOPS ATP SYNTHESIS!!!!!!!!!
SAMPLE QUESTIONS
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Choose the INCORRECT statement concerning the ATP-ADP
cycle and the study of bioenergetics in the human body:
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a. One half of the ATP-ADP cycle involves the coupling the
energy derived from the hydrolysis of the high energy
phosphate bonds of ATP to endergonic reactions so that they
will occur spontaneously.
b. The work that requires energy derived from ATP hydrolysis
includes the transport of electrons down the electron
transport chain.
c. One half of the ATP-ADP cycle involves the generation of
ATP that starts with the formation of reduced coenzymes like
NADH and FADH2and the ultimate transfer of their electrons
to oxygen
d. An important part of oxidative phosphorylation and ATP
biosynthesis is the generation of an electrochemical gradient
across the inner membrane of the mitochondria.
Many catabolic reactions, like the TCA cycle and fatty acid
oxidation, provide the reduced coenzymes for the start of
oxidative phosphorylation and ATP biosynthesis
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• Since electron transport and oxidative phosphorylation
are tightly coupled, which one of the following
mechanisms BEST explains how ADP regulates the rate
of electron transport during oxidative phosphorylation?
• a. AMP concentrations are increased as ADP
concentrations fall
• b. Low [ADP] accelerates the Krebs (TCA) cycle
reaction rates, thereby providing more NADH to activate
electron transport
• c. The transmembrane proton gradient is dissipated
with low [ADP]
• d. The ATP/ADP antiport system is not functional when
mitochondrial [ADP] is low
• e. Proton translocation across the inner mitochondrial
membrane is decreased when ATP-synthase lacks
bound ADP and Pi, secondarily retarding electron
transport
• You isolate mitochondria from a group of patients
that present with lactic acidosis and muscle
weakness, and show that they are unable to: (1)
oxidize reduced coenzyme Q, (2) translocate protons
across their mitochondrial membranes to the
intennembrane space against a concentration
gradient with succinate added as the substrate, and
(3) reduce cytochrome c. The biochemical defect in
these 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)
• Which of the following orderings #1 - #5 of the various components
of the electron transport chain and oxidative phosphorylation will
effectively allow the development of an electrochemical potential
sufficient to drive the generation of high energy phosphate bonds
between ADP and Pi?
• 1. FMN, NADH dehydrogenase, ubiquinone, cytochrome c, cytochrome
oxidase, 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
• As a skilled cell biologist and biochemist, you cleverly
devise a method for experimentally separating the F1
portion of ATP synthase from the membrane-bound Fo
fragment in intact mitochondria. Which of the
following metabolic effects do you observe?
• a. Electron transport and oxygen consumption are
inhibited
• b. Electron transport and phosphorylation of ADP
remain tightly coupled
• c. The inner mitochondrial membrane remains
impermeable to protons
• d. Protons pass through the membrane-bound Fo
fragment, but they do not sustain any ATP formation
• e. The F1 fragment forms ATP at an accelerated rate
until ADP is depleted or the proton gradient is
dissipated
• Which of the following groups of enzymatic reactions,
enzymes and substrates comprise important
anaplerotic pathways for 4-carbon intermediates
critical to the citric acid (TCA) cycle in the liver, muscle
and nervous tissues?
• a. conversion of pyruvate to acetyl CoA via pyruvate
dehydrogenase and glutamate to a-ketoglutarate via
transaminases
• b. conversion of cc-ketoglutarate to glutamate and
GABA
• c. production of ketone bodies (acetoacetate and Phydroxybutyrate)
• d. conversion of pyruvate to oxaloacetate via
pyruvate carboxylase, biotin, bicarbonate ion, and ATP
• e. both (A) and (D)
• Regulation of tricarboxylic acid cycle
activity in vivo may involve the
concentration of all of the following
EXCEPT:
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acetyl CoA
ADP.
ATP.
CoA.
oxygen.
• NAD+ can be regenerated in the cytoplasm if
NADH reacts with any of the following EXCEPT:
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pyruvate.
dihydroxyacetone phosphate.
oxaloacetate.
the flavin bound to NADH dehydrogenase.
phosphoglycerate kinase.
• Glucokinase:
• has a Km considerably greater than the
normal 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.
• A 7yr old female presents with anxiety,
dizziness, sweating and nausea following
brief periods of exercise. The symptoms
are relieved by eating and do not occur if
the patient is frequently fed small meals.
Blood analysis indicates she is
hypoglycemic following brief period of
fasting, 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?
• After the BIOCHEM exam you and your friends
decided to only drink “liquid-fire” (Bacardi 151)
for the rest of the evening. The next morning
you manage to wakeup with terrible ‘hangover’.
Which of these molecules is most responsible
for your hangover?
• Lactic Acid
• Pyruvate
• Acetate
• Acetyladehyde
• Ethanol
• ADH alcohol dehydrogenase
• AcDH acetyladehyde dehydrogenase
• Acetaldehyde forms adducts with Proteins, nucleic acids, and other
compounds results in hangover.
• NADH/NAD+ imbalance causes Liver to over work.
• Diversion of gluconeogenesis by Lactic Acid dehydrogenase decreases
ability of Liver to deliver glucose to the blood.
• In addition, there is increased synthesis of FAT. Acetate + CoA gives you
acetyl-CoA which is a precursor for Fatty acid sythesis. You already have
enough NADH to go to work. So let the FATTYLIVER BEGIN!
HepatoMEGALLY! Lets go!
CLINICAL CORRELATIONS
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Riboflavin Deficiency
– FMN and FAD are both synthesized from riboflavin, which contains the electronaccepting ring structure of FAD
– Severe Riboflavin deficiency decreases the ability of mitochondria to generate ATP
via oxidative phosphorylation
– In general, impairment of Complex I (NADH Dehydrogenase) induces formation of
mitochondria with structural abnormalities.
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Iron Deficiency Anemia
– Characterized by decreased levels of Hb and other heme containing proteins in
blood.
– Iron-containing cytochromes and Fe-S centers of ETC are decreased as well.
– Fatigue partly due to impaired ETC for ATP generation
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ETC inhibitors at specific sites
– Rotenone and Amytal block Complex I
– Antimycin blocks cytochrome b1 in Complex III
– Cyanide blocks cytochrome a/a3 in Complex IV. Prevents reduction of e- from
reduced cytochrome c.
– CO binds to reduced iron of cytochrome oxidase
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Cyanide Poisoning
– CN- causes a rapid and extensive inhibition of ETC at the cytochrome oxidase step.
Prevents O2 from serving as the final e- acceptor.
– Mitochondrial respiration and energy production cease, resulting in cell death
– Occurs from tissue asphyxiation, most notably in the Nervous System
– Treatment: nitrites administered to convert oxyHb to MetheHb, which can then
compete with cytochrome a,a3 for the CN-, forming a complex.
• 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 Oxidative
Phosphorylation. If uncoupled and proton gradient dissipated, ATP and
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-ATPase…ATP synthesis stops.
• Respiration and transport are blocked
• Addition of an uncoupler (DNP) induces initiation of O2
consumption…ETC continues but w/o ATP synthesis since the pathways
are uncoupled.
• Brown Adipose Tissue and Thermogenesis
– Large deposits of brown fat around vital organs (in human
infants)…specialized for ‘non-shivering thermogenesis.’
– Cold or excessive food intake stimulates NE release
– Then Thermogenin, proton conductance uncoupler, is activated,
pumping H+ back into mitochondria…dissipating the gradient.
– ETC is induced, increasing rate of NADH and FADH2 oxidation,
which generates more heat = biological heating pad
•
• Hyperthyroidism – Grave’s Disease
– Thyroid hormone influences bioenergetics via actions on mitochondrial
ox phos.
– In Hyperthyroidism, energy derived from ox. Phos is significantly less
than normal.
– Thryoid causes ‘uncoupling’ of Ox Phos.
– Results in increased heat production – patients complain of feeling hot
and sweaty.
• Salicylate (aspirin) poisoning
– At high concentrations, salicylate can partially uncouple mitochondrial
Ox Phos.
– Decreased ATP [ ] and increased cytosolic AMP induce glycolysis
– Results in increased blood 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, mild
cardiomyopathy
– Maternal inheritance (sex linked)
– Impaired energy metabolism….lactic acidosis
• Pentose Phosphate Pathway
• Hemolysis caused by Reactive Oxygen Species (ROS)
– G6PD deficiency in pentose phosphate pathway
– Causes increased production of radicals from GSH, since can’t produce
sufficient 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 protect
against oxidative stress
– ROS peroxidation of membrane lipids lyses the RBC membrane
• G6PD Mediterranean disease most severe G6PD deficiency
• Lecture 21 – Monosaccharides and interconversion of sugars
• Classical Galactosemia
– Deficiency of Galactosyl-1-P uridylyltransferase
– Accumulation of G-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-Classical Galactosemia
– Galactokinase deficiency
– Unable to convert galactose to galactose-1-P
•
•
Glycogen Synthesis
Glucose Toxicity
– Dysfunction of glycogen synthase
– Due to hyperglycemia…produces insulin resistance
– Due to production of hexosamines that inhibit hexokinase, protein phosphatase 1, and
glycogen synthase.
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Lecture 23 – Glycogen Degradation
Von Gierke’s Disease
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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 diabetes mellitus
– Hyperglycemic
– Continuous glucagon expression causes ketogenesis, lipolysis, and gluconeogenesis.
– Hyperchylomicronemia occurs (liver TG syn and VLDL transport faster than adipose LPL
breakdown 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, particularly
muscles
– Hypertriacylglycerolemia, which results from increase of VLDL without
hyperchylomicronemia. New FA and VLDL synthesized in liver instead of increased
delivery of fatty acids from adipose tissue.
Pentose Phosphate
Pathway
What is the PPP and why is it important?
Pentose Phosphate
Pathway
What is the PPP and why is it important?
The pentose phosphate pathway is primarily an anabolic pathway that utilizes the
6 carbons of glucose to generate 5 carbon sugars and reducing equivalents
Pentose Phosphate Pathway
• To generate reducing equivalents, in the form of NADPH,
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 can
operate to metabolize dietary pentose sugars derived
from the digestion of nucleic acids as well as to
rearrange the carbon skeletons of dietary carbohydrates
into glycolytic/gluconeogenic intermediates
Pentose Phosphate Pathway
• The reactions of fatty acid biosynthesis and steroid biosynthesis
utilize large amounts of NADPH. As a consequence, cells of the liver,
adipose tissue, adrenal cortex, testis and lactating mammary
gland have high levels of the PPP enzymes.
• Erythrocytes utilize the reactions of the PPP to generate large
amounts of NADPH used in the reduction of glutathione
• The conversion of ribonucleotides to deoxyribonucleotides (through
the action of ribonucleotide reductase) requires NADPH as the
electron source, therefore, any rapidly proliferating cell needs
large quantities of NADPH
Pentose Phosphate
Pathway
Oxidative Pathway
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
Reactions catalyzed by glucose-6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase generate one mole of NADPH each for
every mole of glucose-6-phosphate (G6P) that enters the PPP
Pentose Phosphate
Pathway
Non-oxidative Pathway
Non-oxidative reactions are to convert dietary 5 carbon sugars into both 6 (fructose-6phosphate) and 3 (glyceraldehyde-3-phosphate) carbon sugars which can then be
utilized by the pathways of glycolysis
The primary enzymes involved in the non-oxidative steps of the PPP are transaldolase
and transketolase
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 cofactor in the transfer reaction
Transaldolase transfers 3 carbon groups and thus is also involved in a
rearrangement 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
Pentose Phosphate
Pathway
What’s the point?
Pentose Phosphate
Pathway
What’s the point?
R5P production
Oxidation of G6P, a 6 carbon sugar, into a 5 carbon sugar
Generation of NADPH
3 carbon sugar generated is glyceraldehyde-3-phsphate which
can 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)
Pentose Phosphate
Pathway
RBCs
and the PPP
Predominant 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 by glutathione 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 of glutathione peroxidase. The PPP in
erythrocytes is essentially the only pathway for these cells to produce NADPH
Glycogen Metabolism
CH2OH
CH2OH
O
H
H
OH
H
H
OH
H
O
OH
CH2OH
H
H
OH
H
H
OH
H
H
OH
CH2OH
O
H
OH
O
H
OH
H
H
O
O
H
OH
H
H
OH
H
H
O
4
glycogen
H
1
O
6 CH2
5
H
OH
3
H
CH2OH
O
H
2
OH
H
H
1
O
CH2OH
O
H
4 OH
H
H
H
H
O
OH
O
H
OH
H
H
OH
H
OH
Glycogen is a polymer of glucose residues linked by
 a(14) glycosidic bonds, mainly
 a(16) glycosidic bonds, at branch points
Glycogen chains & branches are longer than shown
Glucose is stored as glycogen predominantly in liver and muscle cells.
CH2OH
Glycogen
catabolism
(breakdown):
H
O
H
OH
H
H
OH
OH
H
OPO32
glucose-1-phosphate
Glycogen Phosphorylase catalyzes phosphorolytic
cleavage of the a(14) glycosidic linkages of
glycogen, releasing glucose-1-phosphate as
reaction product.
glycogen(n residues) + Pi 
glycogen (n–1 residues) + glucose-1-phosphate
Commonly used terminology:
 "a" is the form of the enzyme that tends to be active, and
independent of allosteric regulators (in the case of Glycogen
Phosphorylase, when phosphorylated).
 "b" is the form of the enzyme that is dependent on local allosteric
controls (in the case of Glycogen Phosphorylase when
dephosphorylated).
Glycogen catabolism
Most people don’t know…
The relative activity of the un-modified phosphorylase enzyme
(phosphorylase-b) is sufficient to generate enough glucose-1phosphate for entry into glycolysis for the production of
sufficient ATP to maintain the normal resting activity of the
cell; This is true in both liver and muscle cells
Glycogen Phosphorylase in muscle is subject to
allosteric regulation by AMP, ATP, and glucose-6phosphate. A separate isozyme of Phosphorylase
expressed in liver is less sensitive to these allosteric
controls.
 AMP (present significantly when ATP is depleted)
activates Phosphorylase, promoting the relaxed
conformation.
 ATP & glucose-6-phosphate, which both have
binding sites that overlap that of AMP, inhibit
Phosphorylase, promoting the tense conformation.
 Thus glycogen breakdown is inhibited when ATP
and glucose-6-phosphate are plentiful.
Regulation by covalent modification (phosphorylation):
The hormones glucagon and epinephrine activate Gprotein coupled receptors to trigger cAMP cascades.
 Both hormones are produced in response to low
blood sugar.
 Glucagon, which is synthesized by a-cells of the
pancreas, activates cAMP formation in liver.
 Epinephrine activates cAMP formation in muscle.
Glycogen catabolism
•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 of phosphorylase kinase Phosphorylase
kinase activates the enzyme which in turn phosphorylates the b form of
phosphorylase
•Phosphorylation of phosphorylase-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
Hormone (epinephrine or glucagon)
via G Protein (Ga-GTP)
Adenylate cyclase
(inactive)
Adenylate cyclase
(active)
catalysis
ATP
cyclic AMP + PPi
Activation
Signal
cascade by
which
Glycogen
Phosphorylase
is activated.
Phosphodiesterase
AMP
Protein kinase A
(inactive)
Protein kinase A
(active)
ATP
ADP
Phosphorylase kinase
(b-inactive)
Phosphatase
Phosphorylase kinase (P)
(a-active)
ATP
Pi
ADP
Phosphorylase
(b-allosteric)
Phosphorylase (P)
(a-active)
Phosphatase
Pi
The cAMP cascade results in phosphorylation of a serine
hydroxyl of Glycogen Phosphorylase, which promotes transition to
the active (relaxed) state.
The phosphorylated enzyme is less sensitive to allosteric
inhibitors.
Thus, even if cellular ATP & glucose-6-phosphate are high,
Phosphorylase will be active.
The glucose-1-phosphate produced from glycogen in liver may be
converted to free glucose for release to the blood.
With this hormone-activated regulation, the needs of the organism
take precedence over needs of the cell.
Glycogen catabolism
This identical cascade of events occurs in skeletal muscle 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 to phosphorylase 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 utilization
in muscle, the so called fight or flight response
Muscle cells lack glucagon receptors. The presence of glucagon
receptors on muscle cells would be futile anyway since the role of
glucagon release is to increase blood glucose concentrations and
muscle glycogen stores cannot contribute to blood glucose
levels…why?
Glycogen catabolism
– Regulation of phosphorylase kinase activity is also affected by two distinct
mechanisms involving Ca2+ ions
– The ability of Ca2+ ions to regulate phosphorylase kinase is through the
ubiquitous 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 the
neuromuscular junction
– The effect of acetylcholine release from nerve terminals at a neuromuscular
junction is to depolarize the muscle cell leading to increased release of
sarcoplasmic reticulum stored Ca2+, thereby activating phosphorylase
kinase
– Thus, not only does the increased intracellular calcium increase the rate of
muscle contraction it increases glycogenolysis which provides the muscle cell
with the increased ATP it also needs for contraction
Phosphorylase Kinase
inactive
++
Phosphorylase Kinase-Ca
partly active
P-Phosphorylase Kinase-Ca++
fully active
Phosphorylase Kinase in muscle includes calmodulin as its d subunit.
Phosphorylase Kinase is partly activated by binding of Ca++ to this subunit
Phosphorylation of the enzyme, via a cAMP cascade induced by
epinephrine, results in further activation
These regulatory processes ensure release of phosphorylated glucose from
glycogen, for entry into Glycolysis to provide ATP needed for muscle
contraction.
Pyridoxal phosphate (PLP), a
derivative of vitamin B6, serves as
prosthetic group for Glycogen
Phosphorylase.
H
O
O
P
O
O
C
H2
C
OH
O

N
H
CH3
pyridoxal phosphate (PLP)
A class of drugs
developed for treating
the hyperglycemia of
diabetes (chloroindolecarboxamides), inhibit
liver Phosphorylase
allosterically.
These inhibitors bind
at the dimer interface,
stabilizing the inactive
(tense) conformation.
PLP
GlcNAc
inhibitor
GlcNAc
Human Liver
Glycogen Phosphorylase
PLP
PDB 1EM6
Question: Why would an inhibitor of Glycogen
Phosphorylase be a suitable treatment for diabetes?
Debranching enzyme has 2 independent active sites,
consisting of residues in different segments of a single
polypeptide chain:
 The transferase of the debranching enzyme transfers 3
glucose residues from a 4-residue limit branch to the
end of another branch, diminishing the limit branch to a
single glucose residue
 The a(16) glucosidase moiety of the debranching
enzyme then catalyzes hydrolysis of the a(16)
linkage, yielding free glucose. This is a minor fraction of
glucose released from glycogen
 The major product of glycogen breakdown is glucose-1phosphate, from Phosphorylase activity.
Enzyme-Ser-OPO32
CH2OPO32
CH 2OH
H
O
H
OH
H
OH
H
Enzyme-Ser-OPO32
Enzyme-Ser-OH
H
OPO32
OH
glucose-1-phosphate
H
O
H
OH
H
OH
H
OH
CH 2OPO32
H
OPO32
H
O
H
OH
H
H
OH
OH
H
OH
glucose-6-phosphate
Phosphoglucomutase catalyzes this reversible
reaction
Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
The product glucose-6-phosphate may enter Glycolysis or (in liver) be
dephosphorylated for release to the blood
Liver Glucose-6-phosphatase catalyzes the following, essential to the
liver's role in maintaining blood glucose:
glucose-6-phosphate + H2O  glucose + Pi
Most other tissues lack this enzyme…why??
O
CH2OH
Glycogen
synthesis
HN
O
H
H
OH
H
O
H
O
OH
H
OH
UDP-glucose
P
O
O
O
O
P
O
CH2
O
N
O
H
H
OH
H
OH
H
Uridine diphosphate glucose (UDP-glucose) is the
immediate precursor for glycogen synthesis
As glucose residues are added to glycogen, UDP-glucose is
the substrate and UDP is released as a reaction product.
O
UDP-Glucose Pyrophosphorylase
CH2OH
HN
O
H
H
OH
H
O
H
H
O
P
O
OH
O
+

O
O
OH
O
P
O
O
P
O
O
O
O
CH2OH
H
OH
HN
H
O
H
O
OH
H
OH
O
UTP
PPi
O
P
P
O
O
O
O
UDP-glucose
P
O
CH2
O
N
O
H
H
OH
H
OH
H
CH2
O
glucose-1-phosphate
H
O
N
O
H
H
OH
H
OH
H
UDP-glucose is formed from glucose-1-phosphate:
 glucose-1-phosphate + UTP  UDP-glucose + PPi
 PPi + H2O  2 Pi
Overall:
 glucose-1-phosphate + UTP  UDP-glucose + 2 Pi
Spontaneous hydrolysis of the ~P bond in PPi (P~P) drives the overall
reaction
Cleavage of PPi is the only energy cost for glycogen synthesis (one ~P
bond per glucose residue).
Glycogenin initiates glycogen synthesis.
Glycogenin is an enzyme that catalyzes glycosylation of one of its own
tyrosine residues.
6 CH
2OH
H
4
OH
5
O
H
OH
H
H
O
O
C
1
O
3
tyrosine residue
of Glycogenin
UDP-glucose
P
O
P
O
Uridine
HO
C CH
H2
NH
2
H
O
OH
O
6 CH
2OH
O-linked
glucose H
residue 4
OH
5
O
H
OH
H
H
C
1
O
3
2
H
O
OH
C CH
H2
NH
+ UDP
CH OH
CH OH
2
A glycosidic
bond is formed2 between the anomeric C1 of the
O
O
H
H from UDP-glucose
Hglucose moiety
Hderived
and the hydroxyl
H
H
C O
H
oxygen
of
a
tyrosine
side-chain
of
Glycogenin.
H
OH
OH
O
C CH
O a product.
OH
UDP is released as
H
2
H
OH
H
OH
NH
O
6 CH
2OH
O-linked
glucose H
residue 4
5
O
H
OH
OH
UDP-glucose
1
OH
O
H
H
H
OH
H
+ UDP
OH
a(14)
linkage
H
C
H
O
O
OH
H
C CH
H2
NH
2
H
O
CH2OH
O
H
OH
C
O
3
CH2OH
H
H
H
H
OH
C CH
H2
NH
O
+ UDP
Glycosylation at C4 of the O-linked glucose product yields an O-linked
disaccharide with a(14) glycosidic linkage. UDP-glucose is again the
glucose donor
This is repeated until a short linear glucose polymer with a(14)
glycosidic linkages is built up on Glycogenin
Glycogen Synthase catalyzes transfer of the
glucose moiety of UDP-glucose to the hydroxyl at
C4 of the terminal residue of a glycogen chain to
form an a(1 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 of
a glucose residue of glycogen to yield a branch with an
a(16) linkage.
Glycogen Synthesis
UTP UDP + 2 Pi
glycogen(n) + glucose-1-P
glycogen(n + 1)
Glycogen Phosphorylase
Pi
Both synthesis & breakdown of glycogen are spontaneous
If both pathways were active simultaneously in a cell, there would be a "futile
cycle" with cleavage of one ~P bond per cycle (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
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
Glycogen Synthase is allosterically activated by
glucose-6-P
(opposite of effect on Phosphorylase)
Thus Glycogen Synthase is active when high blood glucose leads to
elevated intracellular glucose-6-P
It is useful to a cell to store glucose as glycogen when the input to
Glycolysis (glucose-6-P), and the main product of Glycolysis (ATP), are
adequate.
Glycogen
Glucose-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.
High cytosolic glucose-6-phosphate, which would result when blood
glucose is high, turns off the signal with regard to glycogen synthesis
The conformation of Glycogen Synthase induced by the allosteric
activator glucose-6-phosphate is susceptible to dephosphorylation by
Protein Phosphatase (PP1)
The cAMP cascade induced in liver by glucagon or epinephrine has
the opposite effect on glycogen synthesis.
Glycogen Synthase is phosphorylated by Protein Kinase A as well
as by Phosphorylase Kinase.
Phosphorylation of Glycogen Synthase promotes the "b" (less
active) conformation.
The cAMP cascade thus inhibits glycogen synthesis.
Instead of being converted to glycogen, glucose-1-P in liver may be
converted to glucose-6-P, and dephosphorylated for release to the
blood.
Insulin, produced in response to high blood glucose, triggers a
separate signal cascade that leads to activation of
Phosphoprotein Phosphatase
This phosphatase catalyzes removal of regulatory phosphate
residues from Phosphorylase, Phosphorylase Kinase, &
Glycogen Synthase enzymes
Thus insulin antagonizes effects of the cAMP cascade induced
by glucagon & epinephrine
Glycogen Storage
Diseases are genetic
enzyme deficiencies
associated with excessive
glycogen accumulation
within cells
Some enzymes whose
deficiency leads to glycogen
accumulation are part of the
inter-connected pathways
shown here
glycogen
glucose-1-P
Glucose-6-Phosphatase
glucose-6-P
glucose + Pi
fructose-6-P
Phosphofructokinase
fructose-1,6-bisP
Glycolysis continued
 When an enzyme defect affects mainly glycogen
storage in liver, a common symptom is
hypoglycemia, relating to impaired mobilization
of glucose for release to the blood during fasting.
 When the defect is in muscle tissue, weakness
& difficulty with exercise result from inability to
increase glucose entry into Glycolysis during
exercise.
 Additional symptoms depend on the particular
enzyme that is deficient.
Glycogen Storage Disease
Symptoms, in addition to
glycogen accumulation
Type I, liver deficiency of
Glucose-6-phosphatase (von
Gierke's disease)
hypoglycemia (low blood
glucose) when fasting, liver
enlargement.
Type IV, deficiency of
branching enzyme in various
organs, 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 of
Phosphofructokinase.
inability to exercise.
Summary