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Prior Review
1. pH of strong and weak acids
Stronger acids have a lower pH than weak acids. Weak acids tend to have better buffering propertie
especially when mixed with weak bases.
2. pK, buffers (Henderson-Hasselbach), titration curve
pKa is the acid dissociation constant, pKb is the base dissociation constant. Stronger acids have alarger pKa. As stolen from wikipedia,
if an acid dissociates like HA
Then the
The Henderson-Hasselbalch Equation states that
Namely, the pH of a solution depends on the extent of dissociation of its dissolved acid (a similar
equation exists for basic solutions).
A buffer solution resists change in pH from the addition of acids or bases up to a certain point. Its
usually a mixture of a weak acid and weak base. As more acid is added to the buffer, the weak base
dissociates to raise the pH. When all of the base has dissociated, the buffer breaks. The reverse is
true, of course, when the weak acid dissociates to resist an increase in pH.
3. General Properties of Amino AcidsAn amino acid has a carboxyl (COO-) end, an amino (NH2-) end, and a side chain.
Each side chain has its own pKa. Depending on the pH, this will give the chain a charge. The isoele
point is the pH at which a specific side chain will have no charge. In general, amino acids are assign
a charge based on standard body pH, although there are environments in the body with extremely lo
and high pH.
4. Structure of the peptide bond
The peptide bond links together amino acids from carboxyl end to amino end via dehydration synthe
Its a covalent bond and difficult to break.
Protein Structure and Function
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Learning Objectives
1. General Properties of proteins
Functions: Seen in table below.
Catalysis, Regulation, Transportation, Contractile elements, Defense, Structural elements
Size: molecular weight ranges from 6000 to 40,000,000
Shape: Globular (e.g. hemoglobin, other enzymes), Fibrous (e.g. collagen, contribute to structure),Conjugated (e.g. DNA binding proteins--combine DNA with RNA).
Charge: Depends on amino acids on surface of protein.
Solubilitiy: Depends on location in body. Blood proteins are water soluble, membrane proteins lipid
soluble. Some proteins are amphipathic. Minimum solubility at isoelectric point.
2. Levels of Protein Structure
Primary: Amino acid chain derived directly from gene translation. Each unit is connected via peptide
bonds.
Secondary: Smallest possible structure, connection by weak hydrogen bonds. Most common structu
are the alpha helix and -pleated sheet.
Tertiary: Three-dimensional structure of a single protein chain. Stabilized by hydrogen and ionic bon
Folding usually needs to be done exactly right in order for protein to function properly. Chaperonin
proteins exist to refold damaged proteins correctly, usually by presenting them with a rapidly alterna
hydrophobic and hydrophilic environment. Ribonucleases always fold correctly after denaturation, w
insulin is irreparable when denatured.
Quaternary: The conjugation of multiple tertiary protein structures. Stabilization by hydrogen, ionic, a
hydrophobic interactions. For example, hemoglobin, collagen.
3. Protein folding and denaturation
Discussed above.
4. Structure-function relationships
Protein structure is related to function. Globular proteins usually have hydrophobic center and
hydrophilic surface. Enzymes have a unique active site that binds to a specific substrate. Examples
given in class:
A point mutation on a -chain of hemoglobin results in a long chain instead of a globular protein. The
protein loses its solubility and sickle cell results.
A mutation in CFTR makes it unable to transport Cl- and cystic fibrosis results.
Enzymes August 16, 2011 10:00A1. General properties of enzymes
-Enzymes are a class of proteins that increase the rate of a chemical reaction.
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-Because enzymes control the rates of reactions, they are used to regulate the activity of the c
-Enzymes have a specific distribution within subcellular compartments and within specific
organs.
*As proteins, enzymes are sensitive to changes in temperature and pH and require a relatively
stable environment in order to function.
*Enzymes are often kept in the inactive state, where it is called the zymogen orproenzyme.
This allows enzyme activity to be strictly regulated.
-Many proenzymesrequire a short sequence on the N-terminus to be cleaved in order to beco
active. For example, pepsinogen is translated and released by chief cells in the stomach. Tryp
then cleaves the N-terminus, converting the proenzymeto its active form pepsin.
2. Interaction of enzyme with substrate
-Substrates bind to a relatively small region of an enzyme called the active site. The bound
substrate fits in a specific orientation and is fitted through ionic bonds, hydrogen bonds, and
hydrophobic interactions.-The act of the substrate binding to the enzyme can cause a conformational changein the
enzyme. This is also called induced fit.
3. Enzyme catalysis; Michaelis-Menten equation
-Enzymes have no effect on the thermodynamic properties of a given reaction and therefore
always move the chemical reaction towards equilibrium. Instead, enzymes lower the energy o
activation, an energy barrier required in order for a reaction to proceed, and thereby increase t
speed of a reaction.
-Enzymatic reactions can proceed in the forward or backward reactions depending on where t
chemical equilibrium lies.-Carbonic anhydrase does the reverse and forward reaction depending on location in the body
-The Michaelis-Menten equationallows one to predict the rate of reaction given a specific
amount of substrate.
*During catalysis, the enzyme remains unchanged after the reaction has taken place. In many
cases, the enzyme forms a covalent intermediate. However, this covalent bond is not involved
substrate-enzyme binding.
4. Enzyme inhibition
-Competitive inhibitorsdirectly compete with the substrate to bind at the active site.
-A competitive inhibitor will increase the Km, the concentration required for half the enzyme
be bound to substrate, because the competitive inhibitors will always occupy a specific portio
active enzymes.
-A competitive inhibitor will leave vmax unchanged because adding an infinite amount of
substrate will allow the enzyme to bind to the substrate more often than to the competitor.
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-Noncompetitive inhibitors, also called allosteric inhibitors, bind to a site on the enzymesomewhere other than at the active site.
-Non-competitive inhibitors will occupy a given portion of enzymes at any given time, thereb
reducing vmax regardless of substrate concentration.
*It is hypothesized that the noncompetitive inhibitor binds to the enzyme and prevents it from
achieving a specific conformational state, thereby making the enzyme non-functional.
5. Mechanism of enzyme reactions
-Enzymes can have a high specificity to a given substrate or can be more non-specific (digest
enzymes).
-In a given reaction with an enzyme, two reactants need to bump into each other with the proporientation in order for the reaction to take place. An enzyme binds to these substrates, thereb
increasing effective proximity and placing the substrates into the
proper orientation.
*An enzyme remains unchanged after performing the appropriate chemical reaction.
6. Regulation of enzyme activity
-Isozymesare multiple forms of the same enzyme, often with different kinetic properties.
-Lactate dehydrogenase is given as a specific example where the distribution of lactate
dehydrogenase is specific to different organs.
-Phosphorylation can activate or deactivate a given enzyme.
-Allosteric enzymes
-Multiple subunits can interact with each other or have ligand-induced conformational
change. Binding of first substrate can make second substrate easier to bind
-pH and environment
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*indicates relevant information covered in other lectures but not this one
DNA REPLICATION
8.17.11
Reddy
1. Compare and contrast DNA replication in Prokaryotes and Eukaryotes
Phase Prokaryotes Eukaryotes
Initiation DNA Abinds to OriC(only origin ofreplication) and melts DNAHelicase(?) binds to origin of replication(many)
DNA B(helicase) unwinds DNA Helicase (?) unwinds DNA
Topoisomerase Ineeded to nick 1strand of DNA to relieve torsionalstress (bc continuous circle)
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Single Strand Binding Proteins(SSBs) bind to prevent DNA from re-binding to other parent strand
RPAs bind to prevent DNA from re-binding
to parent strand
Priming Primaserecruited to replication forkand adds RNA primer to leadingstrand, then to lagging strand furtherdown DNA
Primase recruited to replication fork andadds RNA primer to leading strand, thento lagging strand further down DNA
DNA Pol adds a few DNA nucleotides to
primer (part of unit w primase)
Elongation DNA pol IIIthen binds (tethers withbeta clamp) to polymerize DNA in 5-3 direction (leading strand incontinuous manner, and laggingstrand in discontinuous manner wOkazaki fragments)
DNA pol then binds (tethers w PCNA) to
replicate in 5'-3' (leading strand in
continuous manner, and lagging strand in
discontinuous manner w Okazaki
fragments)
DNA Pol III can backtrack andproofread in 3-5 direction
DNA pol can backtrack and proofread in
3'-5' direction
DNA Pol Ireplaces DNA Pol III toremove RNA primers
Fen-1removes primers (bc no polymerase
that has 5-3 exonuclease activity) and
DNA pol replaces gaps w DNA
DNA ligasejoins strands DNA ligasejoins strands
Termination Terminator sequences trapreplication fork near origin site and
bing TUS proteins
Telomerase(type of reverse transcriptase)
creates RNA template to extend lagging
strand with junk DNA
2nd TUS Protein does not allow DNAB to pass through, and elongation isstopped T-loops formed at ends
Topoisomerase ivunlinks thecatenated strands
2. Examples of diseases that occur due to replication defects
a. Mutation in RNA telomerase --> Dyskeratosis congenita: Developmental delay
b. Low telomerase levels --> no T-loops = genomic instability = increased cancer riskc. Fragile X syndromeexcess CGG
d. Muscular dystrophy
e. Spinocerebellar ataxia
f. Huntingtons
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DNA Mutation and Repair Muschen 8.17.2011
Describe the relationship between DNA damage, DNA repair, DNA replication, and mutagenesis
1. MutagenesisPermanent mutation vs Transient alterationa. Mutationerrors during replication, damage induced by chemicals or irradiation;
notconsolidated until next round of replication
b. Transient alterationwhen damage/error reversed by repairc. Depends on the ratio of replication:repairTime frame closes with replicationd.
2. Normal Stem cellsQuiescent (slow cell cycle), High fidelity DNA repair, rare mutations3. Cancer cellsHigh turnover, short life, error-prone DNA repair, mutations drive evolution4. 2nd strand source of correction recruitmentredundancy of dsDNA
State the major sources of DNA damage and the major types of DNA repair1. Sources -
a. Double strand breaks most harmful; complementarity no longer applies, more vulnerato decay/damage
b. Endogenousi.replication errors (misincorporation, slippage) Most frequent, easily repaired
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ii.Deamination (cytosineuracil)iii.Depurination (abasic site (no base) creation)iv.Reactive Oxygen species (strand breaks, base damage)v.DNA recombination errors - Least frequent, difficult to repair
c. Environmentali.IR increasing reactive species (indirect mechanism)
ii.UV generates pyrimidine dimmers (direct mechanism)iii.Chemical Mutagens
2. Repairleast to most seriousa. Proofreadingduring replication. error rate:10^-4 10^-8b. Mismatch excisionafter replication. Error rate 10^-8 10^10
i.Error recognition strand discrimination excision resynthesis ligationc. Base excision repair: DNA glycosylase flips and removes base AP endonuclease
cuts phosphodiester bond DNA polymerase ligasei.creates abasic site that can be premutagenic if not repaired on timeii.Direct reversal: MGMT destroys itself to get rid of methylation of guanine bases
d. Nucleotide excision repair: Damage recognition Nuclease cleavage removal whelicase Pol, Pol DNA ligase
i.removes bulky dimers/unrecognizable basese. Homologous Recombinationless errors, only available during mitosis when sister
chromatid is around. Reliable repair of double strand breaksi.exonuclease cuts to make sticky ends strand invasion by sister chromatid D
synthesis/sister chromatid exchange unwinding/ligation(BLM helicase)f. Non-homologous RecombinationMore error prone, available any time of cell cycle.
Unreliablepossible error in relegation, clean up step is wastefuli.synapse formation to hold ends together by Ku70 and Ku80 DNA PKcs clean
staggered ends Ligation by LIG4 and XRCC4 proteinsDescribe the clinical consequences of mutagenesis and of defects in DNA repair
1. DNA repair deficits Cancer2. Stem cell depletion Premature aging
3. Immune system mutagenesis needed for adaptation to new antigens, etc
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Transcription and Control of Transcription Learning Objectives
1. Describe the basic transcription machinery, the basic structure of genes (including promoters)
and transcription units, and the basic mechanism of transcription in eukaryotes.
a. Basic machinery needed
i. RNA pol(to read your template 3-5)
ii. Some bases(ATP, GTP, CTP, UTP, all ribonucleotides of course)
iii. DNA topoisomerasesto unwind the helix
b. Basic structure of genes, w/promoters & txpn units
ii. Promoter region
1. Where proteins bind to begin transcription. This includes:
2. Initiator sequence(which includes the)
3. TATA box
4. A mix of enhancer and silencer sequences
a. Can be in other places other than right before the transcribed gene (ex.
Behind, in the intron, etc.)
b. Fxn: assist regulation by allowing a specific txpn factor to bind to it
c. This leads to activation/repression of transcription
d. Environmental conditions can control the binding of txpn factors to these
enhancer/silencer elements
e. Ultimately, the binding will lead to actions such as phosphorylation or
binding/dissociation of another protein that is related to the txpn factoriii. Transcribed gene
1. Exon: leaves the nucleus as mature mRNA after modification
2. Intron: Kept inside the nucleus (although problems with the intron could later
contribute with mutations and problems with the mature mRNA)
c. Basic mechanism of euk txpn
i. RNA pol transcribes the DNA
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ii. Depending on the RNA we are attempting to transcribe, we will use a correspondi
polymerase
iii. Basal txpn factors assist pol in recognizing the promoter and initiating txpn
iv. NOTE: Mitochondria (have RNA pol that is similar to prok pol, and transcribes the
own DNA into their own rRNAs, mRNAs, and tRNAs)
2.Discuss the roles of transcriptional activator proteins, enhancer elements, coactivators, and chrom
in regulation of eukaryotic transcription
a. Transcriptional activator proteins
i.Bind basal txpn factors associated with RNA pol 2 to get it over to the promoter
ii. Recruit coactivatorsto perform 2 functions
1. Coactivators are proteins that increase gene expression by binding to a
activator or txpn factor which contains a DNA binding domain, facilitating the txpn of a
desired gene
2. Alter chromatinstructure (like unwind it from the histone) to make
promoter region more accessible
3. Recruit RNA pol II and its basal transcription factors
iii. Enhancer elements(gene sequences far upstream/downstream for the gene
nearby) are brought closer to the gene we want to transcribe through complexes of transcriptional
activator proteins, coactivators, and other transcription factor proteins in preparation for transcript
by RNA pol II
3. Describe the cellular response (or signal transduction) pathway used by steroid hormones and
list the major hormones which interact with members of the nuclear receptor family
a.Major hormones that interact with the steroid receptor protein family also known as thenuclear receptorfamily
i. Glucocorticoids, Mineralcorticoids, Estrogens, Androgens, Progestins
ii. Can also interact with steroid-related vitamins, amino acid derivatives, and other
molecules yet to be discovered
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b. Pathway
i. Steroid comes into the cell and is bound by a steroid receptor
ii. This creates a steroid-protein complex that enters the nucleus (often a dimer),
which binds to a hormone enhancer element on DNA
iii. The bound complex + enhancer sequence will fold up/join the promoter region,
which will now begin to bind txpn factors, coactivators, and Pol II onto the promoter region. TTATA box is illustrated in the example above to give a frame of reference.
iv. Now the desired gene can be transcribed into mRNA
v. The mRNA is then modified and packaged so it can exit the nucleus and be translated
protein
vi. This protein will in turn create a physiological response
4. Explain why agonists promote gene activation by steroid receptors, but antagonists inhibit ste
receptor function
Agonist binding steps Antagonist binding steps
1. Agonist molecule binds to a
receptor. In our notes, the receptor is
a steroid receptor.
1. Antagonist molecule binds to a
receptor
2.Receptor binds to enhancer
sequence on DNA
2.Receptor binds to enhancer
sequence on DNA
3.Receptor undergoes a
conformational change, yielding a
new binding spot
3.Receptor undergoes a
conformational change, BUT there is
NO new binding site
4.A coactivator protein will bind to this
new spot, and with this binding, will
recruit the binding of other
transcriptional factors and RNA Pol 2
4.Coactivator has no place to bind,
the txpn apparatus never sets up
5.Now transcription can occur :) 5.No transcription occurs :*(
Conclusion: promotion of activity Conclusion: inhibited activity
5. Discuss the roles of steroid receptors and their agonist/antagonists in the etiology and/or treatme
breast cancer
a. Breast tissue development is triggered by estrogen
b. Therefore, estrogen is an agonist for breast tissue/breast cancer growth
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c. Tamoxifen, an anti-cancer drug, is an antagonist, changing conformation and inhibiting
txpn by denying coactivators and txpn factors a binding site (see question 4)
d. This prevents the growth of breast cancer cells
6. Explain how the cAMP signaling pathway can regulate txpn of specific genes (Surface cellreceptors)
a. Ex. Glucagon (hormone) pathway (which signals that we need to make glucose)
i. Protein or steroid from outside the cell binds to a receptor.
ii. The receptor activates a G protein, which activates adenylyl cyclase
iii. Adenylyl cyclase releases cAMP, which binds to protein kinase A
iv. Protein kinase A enters the nucleus via nuclear pore, phosphorylating CREB
(cAMP response element binding) protein. Now this is just like question 3!
v. CREB now binds to its enhancer region, CRE (cAMP reponse element)
vi. NOT in the notes, but I thought this was helpful: a coactivator called CBP (CREB
binding protein) then binds to CRE
vii. Now txpn is activated
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---------------------------------------Regulation of Transcription--------------------------------------
Initiation
Can have multiple promoter and start sites
Creates diversity by including/excluding exons
Also changes the UTR length and potential for regulation
Capping
5 end capped by inverted guanine
Some groups are methylated
Done by capping enzymes associated with polymerase as it transcribes
-recognized by nuclear pores, necessary for proper export
-prevents exonuclease degradation
-promotes circularization and translation
PolyadenylationTranscription ends when it recognizes termination sequence AAUAAA
Also can have multiple termination sites-provides 3 end diversity
Once termination sequence is recognized, mRNA is cut 30 bp down and 200 adenosines are
added
Necessary for export of the mRNA
Link to cap to help promote translation
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Prevents 3 end exonuclease degradation
Splicing
Introns out, Exons in
Alternative splicing creates great biodiversity (when intentional)
Temporal/spatial regulation
Consensus site is strong for introns
5 GU..A..C/U rich.AG 3
Excised structure is termed lariat
When accidental or mis-spliced, can be harmful to cell
Dominant negative formsProtein complexes (snRNPs) remain on mRNA, cells can tell if intron is left in
Tend not to be exported
Cryptic sites- sites (sometimes mutations) that become splice acceptor/donor sites that are not
normal sites for splicing
Portuguese family with cystic fibrosis cryptic splice site >> frameshift mutation
Burkitts Lymphoma
chromosomal translocation shortens 3 UTR, removing sequences necessary for mRNA
downregulation
Translation
1) Describe the principle of mRNA translation and explain the degeneracy of genetic code
2) Understand and be able to summarize the general steps of translation
3) Explain how aberrant translation can play a role in human:
Splicing mutations/ frameshift changes
The role of nonsense-mediated mRNA decay
Aminoglycoside antibiotics/deafness
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Carbohydrate Metabolism I, II, III*know the rate-limiters
*know two uses for NADPH (lipid biosynth + reduction of glutathione cross-links in RBC)
*NADH is oxidized to generate ATP, NADPH is oxidized to reduce biomolecules such as glutathione
I. Explain how glucose is metabolized and stored by various tissues in the body.
a. Glucose Sources
Starch and glycogen[ amylase]tri/disaccharides[intestinal lumenenzymes]monosaccharides
Sucrose[intestinal disaccharidase]glucose + fructoseLactose (milk)glucose + galactoseTaken up by intestinal cells that prefer to use glutamine instead of glucose
b. Glucose absorption:
Na dependant co transport: Glucose and GalactoseNa independent co transport: Fructose
c. GLUT (glucose transporters)
GLUT1 RBC, brain
GLUT2 Liver, intestine, kidney, pancreasGLUT3 brain, kidney, placenta
GLUT5 muscle, spermatozoa (prefers fructose)GLUT4 muscle, adipose, heart (insulin-dependant plasma membrane expression)
1. Responds to insulin
2. Stored in vesicles until insulin signaling (blood glucose requires more cellular upta3. Eg: Type I diabetes (insulin secretion deficiency) GLUT4 not expressed on plasma
membrane = hyperglycemia
4. Eg: muscles with defective GLUT4 transporters are weak
d. Tissue Glucose Storage
Tissue Insulin
Response
Glucagon
Response
Glycolysis Acetyl-
CoA
Pentose
Phosphate
Pathway and
NADPH fate
Glycogenesis Other
Liver glycogenesis
glycolysisglycogensis
glycogenolysisgluconeogenesis
glycolysis
Yes Krebs +
OP,
FA
synthesis
Lipid
Biosynthesis
Yes GlycolysisGlycogenesisGycogenolysisGluconeogenesLipid synth (PPPDrug detox
Brain No GLUT4 No GLUT4 Yes Krebs +
OP
Lipid
Biosynthesis
No GlycolysisLipid synth (PPP
RBC No GLUT4 No GLUT4 Yes Lactic
acid (no
mito)
Reduces
Glutathione =
membranes
No GlycolysisLactic acid
fermentation
Muscle
and
Heart
glucoseuptake by
GLUT4
Yes Krebs +
OP
Lipid
Biosynthesis
Yes (not for
body)
Glycolysis
GlycogenesisLipid synth (PPP
Adipose glucoseuptake by
GLUT4
Yes Krebs +
OP,
FA
synthesis
Lipid
Biosynthesis
Yes GlycolysisGlycogenesisLipid synth (PPP
I. Part b: Describe how metabolism of lactose and galactose in individuals can affect fructose intolerance and
galactosemia, respectively.
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Disease Deficiency Stuff that collects Symptoms
Fructose Intolerance Aldolase B: Fructose 1
Phosphate
glyceraldehyde + DHA
Fructose 1 Phosphate ATPPFK1glycolysislacticacid (glycolysis
product)
HypoglycemiaLactic acidosis
Galactosemia Galactose-1-Phosphate
uridyl transferase:
Galactose 1-PUDP-
galactose + Glucose 1-P
Galactose-1-P and
Galactose
Cataracts, mentalretardation
Fail to thrive,vomiting anddiarrhea after milk
ingestion
Lactose Intolerance Lactase: Lactose
Glucose + Galactose
Lactose which feeds happy
gut microbes
The runs
II. Describe how high glucose (hyperglycemia) and low glucose (hypoglycemia) in the circulating blood cause rele
of hormones from pancreas, which affect key enzymes involved in glycolysis, gluconeogenesis and glycogen
synthesis and its breakdown.
Step: Enzyme Insulin Glucagon Other
Controls
cAMP level (phosphatases are
active)
(kinases are active)
Glycolysis
(Glucose glucose-6-phosphate)
Hexokinase - - Inhibited b
G6P
Glycolysis
(Glucose glucose-6-phosphate)
Glucokinase transcription=
ACTIVE
- Liver only
Glycoysis
(fructose-6-phosphate
fructose-1,6-bisphosphate)
PFK1 - - (+) by AMP
F2,6BP
(-) by pH,
citrate, AT
Glycolysis(fructose-6-phosphate
fructose-2,6-bisphosphate)
PFK2 dephosphorylate =ACTIVE (liver)
Phosphorylation=INACTIVE(liver)
Allows insuto indirect
control PF
Glycolysis
(posphoenol
pyruvatepyruvate)
Pyruvate kinase Dephosphorylated:
ACTIVE
Phosphorylation:INACTIVE
Glycogenolysis
Glycogenglucose-1-Glycogen
phosphorylase
Dephosphorylated-
INACTIVE
Phosphorylated-ACTIVE
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phosphate
Glycogenesis
(UDP glucoseglycogen)Glycogen
Synthase
Dephosphorylated-
ACTIVE (A-form)
Phosphorylated-INACTIVE
Gluconeogenesis
(Glucose 6 phosphateglucose)
Glucose-6-
phosphatase
Transcription =ACTIVE Liver andkidney onl
Sucrose is converted to glucose and fructose.
Lactose is converted to glucose and galactose.
Galactose is a monosaccharide.
Other sugars
Fructose goes to fructose-1-phosphate by fructokinase. Aldolase B converts this further. And fructos
metabolites are eventually broken down to pyruvate, which enters the glycolysis pathway.
-A deficiency in aldolase B causes fructose intolerance.
Lactose is converted to glucose and galactose by lactase.
-Galactose has specific enzymes associated with it: Galactose is converted to galactose-1-phosphate by galactokinase.
-Galactose-1-phosphate and UDP glucose are converted to UDP galactose and glucose-1-
phosphate by uridyl transferase. Glucose-1-phosphate is converted to glucose-6-phospate andgoes down the glycolytic pathway.
-Lack of or deficiency of lactase leads to lactose intolerance.
-A deficiency in galactose-1-uridyl transferase leads to galactosemia.
Glycogenesis
Glycogen synthesis occurs in liver and skeletal muscle.10% of the total weight of liver is composed of
glycogen while 1-2% of muscle is composed of
glycogen. Since a person has more muscle than liver,there is a greater absolute amount of glycogen in
muscle.
Glucose is converted to G-6-P by glucokinase (liver)and hexokinase (elsewhere).
G-6-P is converted to G-1-P by mutase.
G-1-P is converted to UDP-glucose by glucose-1-
phosphate uridyltransferase.Glucose can be stored in glycogen using two kinds of
linkages. These are 1,4 and 1,6 linkages.
GlycogenolysisOverall: Glycogen is converted to G-1-P, which is
converted to G-6-P and glucose.
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When blood glucose is low, the liver releases the hormone glucagon. This hormone releases the
secondary messenger cAMP, which activates protein kinase A.-The second messenger can also be activated by the hormone, epinephrine. In the liver, this
causes glucagon breakdown. Since muscle doesnt contain glucagon receptors, glycogen
breakdown occurs through activity of epinephrine. This enables to fight-or-flight response.
When blood glucose is high, the liver releases the hormone insulin. This hormone activates phosphat
activity.
Condition Hormones cAMP Levels Metabolic Process
Fasting Glucagon High Glycogenolysis
Carbohydrates Consumed Insulin Low Glycogenesis
Exercise Epenephrine High Glycogenolysis
Clinical Correlations:
1) Patient has abnormally enlarged liver and hypoglycemia is observed far more often than expected.The patient is tested for deficiency in liver enzymes. What are your two differential diagnoses?
Answer: Deficiency in glucose-6-phosphatase (von Geirkes disease) or a deficiency in liver-specific
glycogen phosphorylase would explain the symptoms.
2) Patient has a sugary meal but soon starts to feel sick. A blood test reveals the patient to havehypoglycemia, lactic acidosis, and increased hemolysis. Also, intracellular ATP is reduced. Explain t
diagnosis and the symptoms.
Diagnosis: The patient has fructose intolerance. Lack of aldolase B causes an accumulation of fructos1-phosphate (substrate for aldolase B). Fructose-1-phosphate sequesters free phosphate, which preven
the formation of ATP. Low intracellular ATP is a positive regulator of phosphfructokinase-1, which
stimulates glycolysis and explains symptoms.
3) Two infants at the same hospital show a poor response to milk. Both infants have diarrhea after bu
one also presents with an enlarged liver, vomiting, and a failure to thrive. What do these infants have
and what are their prognoses?
Diagnosis: The first infant has lactose intolerance. It is non-lethal and easy to correct with dietarychanges. The second infant has galactosemia, which is far more serious. In the avsence of an enzyme
galactose is converted to galacitol which leads to cataract formation. Toxic effects are lessened whenmilk is minimized in the diet but the infant will still show long-term complications including mental
retardation.
4) Patient has exercise-induced muscular pain as well as cramps and progressive hypoglycemia. Live
normal. What enzyme deficiency would explain this?
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Diagnosis: A deficiency in muscle-specific glycogen phosphorylase would explain symptoms as wel
why the liver is unaffected.
Gluconeogenesis: Creation of glucose from lactate occurs in Liver
- also in Kidney under starving conditions- occurs 18-24 hours after eating, glycogen stores are depleted-
Precursors:- Lactate- Alanine: converted to Pyruvat- Glycerol
Note which Enzymes are different than Glycolysis
Glycolyis Gluconeogenesis
Hexokinase/ Glucokinase G-6-Phosphatase
Phosphofructokinase-1 Fructose-1,6-Bisphosphatase
Pyruvate Kinase PEP carboxykinase
Pyruvate Carboxylase
5. Explain how a genetic deficiency of glucose-6-phospate dehydrogenase in RBCs leads to
hemolytic anemia
G-6-P Dehydrogenase converts:
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This reaction generates NADPH as it reduces the NADP+ cofactor.
- NADPH is a cofactor in reducing GSSG GSG
- GSSG = oxidized glutathione
- GSH = Glutathione (reduced)
-GSH oxidizes to GSSG to break cross-linking of sulphidryl (-SH) groups
- A reduction GSH will result in increased cross-linking, leading to rigid blood vessels which lyse eas
in capillary beds and the pulp of the spleen.
- Oxidant drugs dramatically exacerbate this problem
6. Describe how hyperglycemic conditions generate glucose-protein adducts (AGE) which ar
deleterious to cells
AGE formation is due to prolonged high blood glucose levels exposed to hemoglobin molecules. AG
binds to RAGE (AGE receptor) resulting in the release of chemokines and cytokines. These cause
monocytes to transmigrate across the arterial wall and uptake oxidized LDL. These monocytes beco
Foam Cellsand cause inflammation and atherosclerosis (thickening of the wall) in the artery.
7. Explain how AGE molecules (HbA) are used as a metabolic index of diabetes control
AGE (advanced glycation end products) are covalent linkages between glucose and proteins. The
adducts form without enzymes through non-enzymatic glyocsylation. The amount of adducts form
is directly proportional to the glucose concentrationand duration of exposure to
macromolecules (specifically Hemoglobin). Higher concentrations of HbA1cindicate a long-termhyperglycemia in the blood. Because HbA1cconcentrations are not immediately susceptible to chang
in blood glucose levels, they provide a gauge of glucose levels that isnt affected by prior food
consumption (as opposed to insulin levels, or blood glucose levels).
Example: After fasting, a diabetic could have a glucose lvl of 150 mg/dL but a HbA1cof 7.8%
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Bioenergetics and Oxidative Metabolism IObjectives
1. Role of the ATP cycle in anabolic and catabolic pathways:
Catabolic reactions generate ATP by oxidation: carbs, fats, aa Anabolic reactions utilize ATP in the synthesisof macromolecules, muscle contraction, ac
transport, nerve conduction and thermogenesis.
High energy bond in ATP = phosphoanhydride bond between gamma and beta carbons2. Name the three general classes of substances that are oxidized in order to from ATP
Carbohydrates (Glycogenolysisglucosepyruvate) Fats (Lipolysisfatty acidsacetyl CoA) Proteins (Proteinolysisamino acidspyruvate, acetyl CoA)
3. Write an equation relating Gibbs free energy (G) to enthalpy (H) and entropy (S). Describe howchanges in G are related to exergonic and endergonic reactions and to equilibrium
G = H - TS Exergonic reaction: G < 0 Endergonic reaction: G > 0
At equilibrium G = 04. Explain the importance of pyruvate dehydrogenase (PDH) in oxidative metabolism and describe
regulation. Name the five cofactors utilized by this enzyme.
Pyruvate: alpha-keto carboxylic acid, glucogenic, decarboxylatedacetyl CoA + CO2 Pyruvate dehydrogenase functions: Krebs cycle, FA synthesis, FA oxidation, ketone body
synthesis and oxidation, cholesterol synthesis, aa, FA metabolism
PDH in oxidative metabolism: pyruvate is transported across inner mitochondrial membrinto the matrix where it is oxidized by PDH to acetyl CoA
PDH structure: 3 catalytic subunits (E1, E2, E3), 2 regulator subunits, one binding proteiThree subunits pass the substrate along to complete the whole reaction.
Regulation: (+) Mg2+, Ca2+, (-) PD products, NADH, Acetyl CoA Indirect Feedback Regulation: lipoyl-lysine binds pyruvate dehydrogenase kinase 3 (PDK
stimulates kinase, phosphorylates PDH E1, inactivates enzyme
PDH cofactors:i. Coenzyme A
ii. NAD (nicotinamide adenine dinucleotide)iii. FAD (flavin adenine dinucleotide)iv. TPP (thiamine pyrophosphate) *vitamin B deficiency = Beriberiv. Lip (Lipoic acid)
5. Name the most important function of the Krebs cycle and list three other functions.
Production of ATP Acetyl CoA is oxidized to 2 molecules of CO2, CoA released ?
6. Identify three energy-rich products produced by the Krebs cycle and discuss their role inbioenergetics.
NADH (electron carrier, 2e-, 1H+) enters ETC, needed for ATP production FADH2 (e- carrier) enters ETC, needed for ATP production GTP )= ATP (cellular energy)
7. Recognize the names of the enzymes and intermediates in the Krebs cycle.
Citrate Isocitrate
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Ketoglutarate Succinyl-CoA Succinate Fumarate Malate
Oxaloacetate8. Briefly describe how the Krebs cycle intermediates are generated. Regulation based on availability of substrates, availability of O2, need for energy (ATP), a
allosteric enzyme regulation
Krebs cycle: delta G
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Lipid Metabolism I (synthesis)Describe the general structure of fatty acids and discuss where and when they are made.
HOOC-hydrocarbon tailProperties:
-Fatty acids are ionized at physiological pH which makes them charged and amphipathic.
-Naturally occurring fatty acids have an even number of carbon atoms (synthesized two Cs a
time).-Saturated, monounsaturated (monoenoic), or polyunsaturated (polyenoic)
Recognize the names of common fatty acids and the two essential fatty acids.Common fatty acids: palmitic acid, palmitoleic acid.
Essential fatty acids: linolenic acid, linoleic acid.
Name the precursors of the fatty acid synthesis.
Acetyl CoA
-Acetyl CoA is derived from pyruvate in the mitochondria by pyruvate dehydrogenase. Howeacetyl CoA cannot cross the membrane into the cytoplasm. To overcome this, acetyl CoA is
destroyed in the mitochondria and generated in the cytoplasm using the citrate shuttle (think
transporter from Star Trek).Malonyl CoA
-Malonyl CoA is a substrate of fatty acid synthesis. It is generated from acetyl CoA by acetyl
CoA carboxylase. The carboxylic group comes from bicarbonate.
Name the two enzyme complexes responsible for fatty acid synthesis and identify their
intracellular location.Acetyl CoA carboxylase is located in the cytoplasm.
Fatty Acid Synthase is located in the cytoplasm.
Discuss the regulation of fatty acid synthesis.
Fats are made when sugars are present, which means insulin is present. Since insulin activates
dephosphorylase activity, enzymes in the fatty acid synthesis pathway are typically activated in theunphosphorylated state. Likewise, the presence of glucagon suppresses fatty acid synthesis because o
its activation of kinases.
Citrate activates acetyl CoA carboxylase, the rate-limiting step of fatty acid synthesis.-Citrate is used in the Krebs cycle. Its presence indicated a well-fed state.
Describe how fatty acids are made longer and how double bonds are generated.
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Acetyl CoA is the two carbon building block that starts fatty acid synthesis. Malonyl CoA is added to
acetyl CoA in a 2+3=4 fashion. Then another malonyl CoA is added to the 4-carbon intermediate in a
4+3=6 fashion. This continues until a 16-carbon fatty acid (palmitic acid) is generated. The extracarbons are lost as CO2.
The entire process takes place in a large enzyme complex called fatty acid synthase. Each time malonCoA is added to the intermediate, one cycle through fatty acid synthase has occurred. Generating onefatty acid requires 7 cycles and 1 acetyl CoA, 7 malonyl CoA, ATP, and 14 NADPH (produced in
pentose pathway). The pathway reduces malonyl CoA twice (C=O becomes CH2).
Describe the general structure of triacylglycerols and discuss where and when they are made.
Fatty acids are synthesized in the liver and intestine (mostly liver) but are stored as triglycerides in
adipose tissue and muscle (mostly adipose). They are transported through the blood as very low densliposomes (VLDLs).
Stuff thats not covered under the learning objectives but are probably important .Peroxisomes subject very long chain fatty acids to beta oxidation until they are short enough (rule of
thumb, 18 carbons or less). They are then transported to the mitochondria where they are broken dow
for energy.
Carboxylation reactions require a biotin cofactor. In this reaction, that means acetyl CoA carboxylase
People who eat raw eggs are at risk for biotin deficiency.
Pantothenic acid is a cofactor required for fatty acid synthase. Only alcoholics are at risk for pantothe
acid deficiency.
Fatty acid synthase makes palmitic acid, which is a 16-carbon saturated fatty acid. This one product g
on to make families of other products. In the cytoplasm, elongase activity adds malonyl CoA in a
16+3=18 fashion, similar to that used by fatty acid synthase. In the mitochondria, acetyl CoA is addedirectly (even numbered fatty acids only). Desaturase enzymes add in double bonds at specific locati
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Lipid Metabolism II (Mobilization and Oxidation)
Remember:
Insulin phosphatase (Dephosphorylation): protein that are active when dephosphorylated areanabolic (ex. PFK-1, Glycogen Synthase)
Glutagon kinase (Phosphorylation): proteins that are active when phosphorylated are catabolic
G-6-Pase, Glycogen Phosporylase)
1. Discuss when and how fats are mobil ized from adipose tissue
Fats are stored as TAG (Triacylglycerol) and needs to be converted to FFA (Free Fatty Acid) before
entering the blood stream.
I. Conversion TAG FFA
a. FAs are removed stepwise TAG -> DAG -> MAG Glycerol
b. TAG
Diacyglycerol : catalyzed by Hormone Sensitive Lipase (HSL)i. HSL is the rate limiting step
ii. HSL binds to Perilipin Ain a phosphorylated state
iii. Perlipin binds lipid drops and is inactive when dephosphorylated
c. Fast lipases catalyze later steps
II. Fat mobilization:
a. low insulin levels stimulate fat mobilizationb. Transported in the blood bound to albumin
2. Describe how free (unesteri f ied) fatty acids are transported in the b lood.
Free Fatty Acids are transported in the blood bound to Albumin. If the FFAs were unbound they wo
disrupt Cell Plasma Membranes in the blood vessels
3. Identi fy t issues where fatty acids o xidation occu rs
- Liver: can synthesize ketone bodies
- Muscle: solely used for energy
4. Describe -oxidation and discu ss how energy is generated from this pathw ay. Where and
when does this happen?
Where:
-oxidation occurs in the mitochondria and peroxisomes of Muscle and Liver Tissue (sometimes
kidney)
- FFA FA-Coa (ester) occurs in cytoplasm
Mitochondria:
- CPT I/II(Carnitine- Palmitoyl-acytransferase)and Translocasetransport FA-CoA intomitochondrial lumen.
o Carnitineis synthesized or provided by diet Synthesis requires Vit C
- C12 or less can pass through transporterPeroxisomes:
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- Shortens FAs to < C8, then transported to mitochondria- Produces No ATP- Generates Acetyl Coa
-oxidation
-Basic: Acyl-Coa + Coa Acyl-Coa (smaller) + Acetyl Coa
- 4 steps per Acetyl Coa production
-Palmitoyl CoA + 7 CoA + 7 FAD + 7 NAD + 7 H2O8Acetyl CoA+ 7 FADH2+ 7 NADH
-Cofactors: FAD, NAD, H20 FADH2+ NADH
-Catalyzed by Mitochondrial Trifunctional protein (MTF): Steps 2-4
- 108 ATP per Palmitic Acid (C:16)
5. Name the three ketone bodies and disc uss w hen and w here they are made and u ti l ized
- Ketone synthesis occurs from -oxidation: fasting, starvation, diabetes
- Ketone bodiesAcetoacetate, -hydroxybutyrate, Acetone
- Ketones utilized via Ketone Body oxidation in the brain during fasting (50% of energy)
-prevents protein breakdown: AA glucose
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6. Describe events that occur during starvation or in untreated diabetes when excess ketone
bodies are formed.Ketoacidosis results from high levels of ketone (conjugate bases) in the blood.
- mechanism prevents catabolism of muscle in the short term
(Optional) Explain how b ears hibernate for month s witho ut gett ing dehydrated. How d o came
make it throug h the desert?
Camels and Bears both use stores of fatty acids to survive. They are a more efficient store of energy
that Carbohydrates (9 Kcal/g vs 1Kcal/g [in water]).
(Optional) Give the probable cause of death for an anorexic patient and explain your
reasoning.
Anorexics have no fat stores so catabolize protein for energy. They die from heart failure due to mus
mass reducation in the heart