biochem & mol bio

8/13/2019 Biochem & Mol Bio

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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

biochem & mol bio

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