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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 properties, 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 a larger 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. It‟s 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 Acids An 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 isoelectric point is the pH at which a specific side chain will have no charge. In general, amino acids are assigned a charge based on standard body pH, although there are environments in the body with extremely low and high pH. 4. Structure of the peptide bond The peptide bond links together amino acids from carboxyl end to amino end via dehydration synthesis. It‟s a covalent bond and difficult to break.

Protein Structure and Function

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 structures are the alpha helix and β-pleated sheet. Tertiary: Three-dimensional structure of a single protein chain. Stabilized by hydrogen and ionic bonds. 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 alternating hydrophobic and hydrophilic environment. Ribonucleases always fold correctly after denaturation, while insulin is irreparable when denatured. Quaternary: The conjugation of multiple tertiary protein structures. Stabilization by hydrogen, ionic, and 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 β-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 10:00AM

1. General properties of enzymes -Enzymes are a class of proteins that increase the rate of a chemical reaction.

-Because enzymes control the rates of reactions, they are used to regulate the activity of the cell. -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 or proenzyme. This allows enzyme activity to be strictly regulated. -Many proenzymes require a short sequence on the N-terminus to be cleaved in order to become active. For example, pepsinogen is translated and released by chief cells in the stomach. Trypsin then cleaves the N-terminus, converting the proenzyme to 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 change in 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 of activation, an energy barrier required in order for a reaction to proceed, and thereby increase the speed of a reaction. -Enzymatic reactions can proceed in the forward or backward reactions depending on where the chemical equilibrium lies. -Carbonic anhydrase does the reverse and forward reaction depending on location in the body. -The Michaelis-Menten equation allows 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 in substrate-enzyme binding. 4. Enzyme inhibition -Competitive inhibitors directly compete with the substrate to bind at the active site. -A competitive inhibitor will increase the Km, the concentration required for half the enzymes to be bound to substrate, because the competitive inhibitors will always occupy a specific portion of 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.

-Noncompetitive inhibitors, also called allosteric inhibitors, bind to a site on the enzyme somewhere other than at the active site. -Non-competitive inhibitors will occupy a given portion of enzymes at any given time, thereby 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 (digestive enzymes). -In a given reaction with an enzyme, two reactants need to bump into each other with the proper orientation in order for the reaction to take place. An enzyme binds to these substrates, thereby 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 -Isozymes are 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

*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 Initiation Prokaryotes DNA A binds to OriC (only origin of replication) and melts DNA DNA B (helicase) unwinds DNA Topoisomerase I needed to nick 1 strand of DNA to relieve torsional stress (bc continuous circle) Eukaryotes Helicase(?) binds to origin of replication (many) Helicase (?) unwinds DNA

Single Strand Binding Proteins (SSBs) bind to prevent DNA from rebinding to other parent strand Priming Primase recruited to replication fork and adds RNA primer to leading strand, then to lagging strand further down DNA

RPAs bind to prevent DNA from re-binding to parent strand Primase recruited to replication fork and adds RNA primer to leading strand, then to lagging strand further down DNA DNA Pol α adds a few DNA nucleotides to primer (part of unit w primase)

Elongation

DNA pol III then binds (tethers with beta clamp) to polymerize DNA in 5‟3‟ direction (leading strand in continuous manner, and lagging strand in discontinuous manner w Okazaki fragments) DNA Pol III can backtrack and proofread in 3‟-5‟ direction DNA Pol I replaces DNA Pol III to remove RNA primers

DNA pol δ 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 δ can backtrack and proofread in 3'-5' direction Fen-1 removes primers (bc no polymerase that has 5’-3’ exonuclease activity) and DNA pol δ replaces gaps w DNA DNA ligase joins strands Telomerase (type of reverse transcriptase) creates RNA template to extend lagging strand with junk DNA

DNA ligase joins strands Termination Terminator sequences trap replication fork near origin site and bing TUS proteins 2nd TUS Protein does not allow DNA B to pass through, and elongation is stopped Topoisomerase iv unlinks the catenated strands 2.

T-loops formed at ends

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 risk c. Fragile X syndrome – excess CGG d. Muscular dystrophy e. Spinocerebellar ataxia f. Huntington‟s

DNA Mutation and Repair – Muschen 8.17.2011 Describe the relationship between DNA damage, DNA repair, DNA replication, and mutagenesis 1. Mutagenesis – Permanent mutation vs Transient alteration a. Mutation – errors during replication, damage induced by chemicals or irradiation;

not consolidated until next round of replication b. Transient alteration – when damage/error reversed by repair c. Depends on the ratio of replication:repair – Time frame closes with replication d. 2. Normal Stem cells – Quiescent (slow cell cycle), High fidelity DNA repair, rare mutations 3. Cancer cells – High turnover, short life, error-prone DNA repair, mutations drive evolution 4. 2nd strand source of correction recruitment – redundancy of dsDNA State the major sources of DNA damage and the major types of DNA repair 1. Sources a. Double strand breaks most harmful; complementarity no longer applies, more vulnerable to decay/damage b. Endogenous i.replication errors (misincorporation, slippage) Most frequent, easily repaired

ii.Deamination (cytosine – uracil) 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. Environmental i.IR increasing reactive species (indirect mechanism) ii.UV generates pyrimidine dimmers (direct mechanism) iii.Chemical Mutagens 2. Repair – least to most serious a. Proofreading – during replication. error rate:10^-4 → 10^-8 b. Mismatch excision – after replication. Error rate 10^-8 → 10^10 i.Error recognition → strand discrimination → excision → resynthesis → ligation c. Base excision repair: DNA glycosylase flips and removes base → AP endonuclease cuts phosphodiester bond → DNA polymerase → ligase i.creates abasic site that can be premutagenic if not repaired on time ii.Direct reversal: MGMT destroys itself to get rid of methylation of guanine bases d. Nucleotide excision repair: Damage recognition → Nuclease cleavage → removal with helicase → Polδ, Polε → DNA ligase i.removes bulky dimers/unrecognizable bases e. Homologous Recombination – less errors, only available during mitosis when sister chromatid is around. Reliable repair of double strand breaks i.exonuclease cuts to make sticky ends → strand invasion by sister chromatid → DNA synthesis/sister chromatid exchange → unwinding/ligation(BLM helicase) f. Non-homologous Recombination – More error prone, available any time of cell cycle. Unreliable – possible error in relegation, clean up step is wasteful i.synapse formation to hold ends together by Ku70 and Ku80 → DNA PKcs clean up staggered ends → Ligation by LIG4 and XRCC4 proteins Describe the clinical consequences of mutagenesis and of defects in DNA repair 1. DNA repair deficits → Cancer 2. Stem cell depletion → Premature aging 3. Immune system → mutagenesis needed for adaptation to new antigens, etc

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 topoisomerases to unwind the helix b. Basic structure of genes, w/promoters & txpn units

ii.

c.

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 factor iii. 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) Basic mechanism of euk txpn i. RNA pol transcribes the DNA

ii. polymerase

Depending on the RNA we are attempting to transcribe, we will use a corresponding

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 their own DNA into their own rRNAs, mRNAs, and tRNAs)

2.Discuss the roles of transcriptional activator proteins, enhancer elements, coactivators, and chromatin 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 coactivators to perform 2 functions 1. Coactivators are proteins that increase gene expression by binding to an activator or txpn factor which contains a DNA binding domain, facilitating the txpn of a desired gene 2. Alter chromatin structure (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 or 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 transcription 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 the „nuclear receptor‟ family i. Glucocorticoids, Mineralcorticoids, Estrogens, Androgens, Progestins ii. Can also interact with steroid-related vitamins, amino acid derivatives, and other molecules yet to be discovered

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. The TATA 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 into protein vi. This protein will in turn create a physiological response

4. Explain why agonists promote gene activation by steroid receptors, but antagonists inhibit steroid receptor function Agonist binding steps Antagonist binding steps

1. Agonist molecule binds to a receptor. In our notes, the receptor is a steroid receptor. 2.Receptor binds to enhancer sequence on DNA

1. Antagonist molecule binds to a receptor

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 5.Now transcription can occur :)

4.Coactivator has no place to bind, the txpn apparatus never sets up

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 treatment of breast cancer a. Breast tissue development is triggered by estrogen b. Therefore, estrogen is an agonist for breast tissue/breast cancer growth

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 cell receptors) 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 (CREBbinding protein) then binds to CRE vii. Now txpn is activated

---------------------------------------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 Polyadenylation Transcription 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

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 forms Protein 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 the normal sites for splicing
Portuguese family with cystic fibrosis cryptic splice site >> frameshift mutation Burkitt’s 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

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 [α amylase]tri/disaccharides[intestinal lumen enzymes]monosaccharides  Sucrose [intestinal disaccharidase]glucose + fructose  Lactose (milk)glucose + galactose  Taken up by intestinal cells that prefer to use glutamine instead of glucose b. Glucose absorption:  Na dependant co transport: Glucose and Galactose  Na independent co transport: Fructose c. GLUT (glucose transporters)  GLUT1 – RBC, brain  GLUT2 – Liver, intestine, kidney, pancreas  GLUT3 – 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 uptake) 3. 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 AcetylCoA Pentose Phosphate Pathway and NADPH fate Lipid Biosynthesis Glycogenesis Other

Liver

glycogenesis glycolysis

glycogensis glycogenolysis gluconeogenesis glycolysis No GLUT4 No GLUT4

Yes

Krebs + OP, FA synthesis

Yes

Brain RBC

No GLUT4 No GLUT4

Yes Yes

Muscle and Heart Adipose

glucose uptake by GLUT4 glucose uptake by GLUT4

Yes

Krebs + OP Lactic acid (no mito) Krebs + OP Krebs + OP, FA synthesis

Lipid Biosynthesis Reduces Glutathione =  membranes Lipid Biosynthesis Lipid Biosynthesis

No No

       

Glycolysis Glycogenesis Gycogenolysis Gluconeogenesis Lipid synth (PPP) Drug detox Glycolysis Lipid synth (PPP)

 Glycolysis  Lactic acid fermentation  Glycolysis  Glycogenesis  Lipid synth (PPP)  Glycolysis  Glycogenesis  Lipid synth (PPP)

Yes (not for body) Yes

Yes

I.

Part b: Describe how metabolism of lactose and galactose in individuals can affect fructose intolerance and galactosemia, respectively.

Disease Fructose Intolerance

Deficiency Aldolase B: Fructose 1 Phosphate glyceraldehyde + DHA

Stuff that collects Fructose 1 Phosphate

Symptoms  ATPPFK1 glycolysis lactic acid (glycolysis product)  Hypoglycemia  Lactic acidosis

Galactosemia

Galactose-1-Phosphate uridyl transferase: Galactose 1-P UDPgalactose + Glucose 1-P

Galactose-1-P and Galactose

 Cataracts, mental retardation  Fail to thrive, vomiting and diarrhea 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 release of hormones from pancreas, which affect key enzymes involved in glycolysis, gluconeogenesis and glycogen synthesis and its breakdown. Enzyme Insulin Glucagon Other Controls

Step:

cAMP level Glycolysis (Glucose glucose-6phosphate) Glycolysis (Glucose glucose-6phosphate) Glycoysis (fructose-6-phosphate  fructose-1,6-bisphosphate) Hexokinase

↓ (phosphatases are active) -

↑ (kinases are active) Inhibited by G6P Liver only

Glucokinase

↑ transcription= ACTIVE -

-

PFK1

-

(+) by AMP & F2,6BP (-) by ↓pH, citrate, ATP Allows insulin to indirectly control PFK1

Glycolysis (fructose-6-phosphate  fructose-2,6-bisphosphate) Glycolysis (posphoenol pyruvatepyruvate) Glycogenolysis Glycogenglucose-1-

PFK2

dephosphorylate = ACTIVE (liver)

Phosphorylation=INACTIVE (liver)

Pyruvate kinase

Dephosphorylated: ACTIVE DephosphorylatedINACTIVE

Phosphorylation:INACTIVE

Glycogen phosphorylase

Phosphorylated-ACTIVE

phosphate Glycogenesis (UDP glucoseglycogen) Gluconeogenesis (Glucose 6 phosphate glucose)

Glycogen Synthase Glucose-6phosphatase

DephosphorylatedACTIVE (A-form)

Phosphorylated-INACTIVE Transcription =ACTIVE Liver and kidney only

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 fructose 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-1phosphate by galactokinase. -Galactose-1-phosphate and UDP glucose are converted to UDP galactose and glucose-1phosphate by uridyl transferase. Glucose-1-phosphate is converted to glucose-6-phospate and goes 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-1phosphate uridyltransferase. Glucose can be stored in glycogen using two kinds of linkages. These are 1,4 and 1,6 linkages.

Glycogenolysis Overall: Glycogen is converted to G-1-P, which is converted to G-6-P and glucose.

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 doesn’t 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 phosphatase activity. Condition Fasting Carbohydrates Consumed Exercise Hormones Glucagon Insulin Epenephrine cAMP Levels High Low High Metabolic Process Glycogenolysis Glycogenesis 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 Geirke’s 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 have hypoglycemia, lactic acidosis, and increased hemolysis. Also, intracellular ATP is reduced. Explain the diagnosis and the symptoms. Diagnosis: The patient has fructose intolerance. Lack of aldolase B causes an accumulation of fructose1-phosphate (substrate for aldolase B). Fructose-1-phosphate sequesters free phosphate, which prevents 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 but 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 dietary changes. 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 when milk 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. Liver is normal. What enzyme deficiency would explain this?

Diagnosis: A deficiency in muscle-specific glycogen phosphorylase would explain symptoms as well as 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:

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 easily 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 are deleterious to cells AGE formation is due to prolonged high blood glucose levels exposed to hemoglobin molecules. AGE 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 become Foam Cells and 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 formed is directly proportional to the glucose concentration and duration of exposure to macromolecules (specifically Hemoglobin). Higher concentrations of HbA1c indicate a long-term hyperglycemia in the blood. Because HbA1c concentrations are not immediately susceptible to changes in blood glucose levels, they provide a gauge of glucose levels that isn‟t 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 HbA1c of 7.8%

Bioenergetics and Oxidative Metabolism I
Objectives 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 synthesis of macromolecules, muscle contraction, active transport, nerve conduction and thermogenesis.  “High energy” bond in ATP = phosphoanhydride bond between gamma and beta carbons 2. Name the three general classes of substances that are oxidized in order to from ATP  Carbohydrates (Glycogenolysis  glucose  pyruvate)  Fats (Lipolysis  fatty acids  acetyl CoA)  Proteins (Proteinolysis  amino acids  pyruvate, acetyl CoA) 3. Write an equation relating Gibbs free energy (G) to enthalpy (H) and entropy (S). Describe how changes in G are related to exergonic and endergonic reactions and to equilibrium  G = H - TS  Exergonic reaction: G < 0  Endergonic reaction: G > 0  At equilibrium G = 0 4. Explain the importance of pyruvate dehydrogenase (PDH) in oxidative metabolism and describe its regulation. Name the five cofactors utilized by this enzyme.  Pyruvate: alpha-keto carboxylic acid, glucogenic, decarboxylated  acetyl 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 membrane into the matrix where it is oxidized by PDH to acetyl CoA  PDH structure: 3 catalytic subunits (E1, E2, E3), 2 regulator subunits, one binding protein. Three 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 (PDK3), 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 = Beriberi v. 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 in bioenergetics.  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

 Ketoglutarate  Succinyl-CoA  Succinate  Fumarate  Malate  Oxaloacetate 8. Briefly describe how the Krebs cycle intermediates are generated.  Regulation based on availability of substrates, availability of O2, need for energy (ATP), and allosteric enzyme regulation  Krebs cycle: delta G << 0, highly exergonic  ? 9. Name the intracellular location of the Krebs cycle and oxidative metabolism.  Kreb Cycle: mitochondrial matrix  Oxidative metabolism: inner mitochondrial membrane

Lipid Metabolism I (synthesis)
Describe the general structure of fatty acids and discuss where and when they are made. HOOC-hydrocarbon tail Properties: -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 C’s at 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. However, acetyl 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 the unphosphorylated state. Likewise, the presence of glucagon suppresses fatty acid synthesis because of 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.

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 extra carbons are lost as CO2. The entire process takes place in a large enzyme complex called fatty acid synthase. Each time malonyl CoA is added to the intermediate, one cycle through fatty acid synthase has occurred. Generating one fatty 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 density liposomes (VLDL’s). Stuff that’s 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 down 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 pantothenic acid deficiency. Fatty acid synthase makes palmitic acid, which is a 16-carbon saturated fatty acid. This one product goes 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 added directly (even numbered fatty acids only). Desaturase enzymes add in double bonds at specific locations.

Lipid Metabolism II (Mobilization and Oxidation)
Remember: Insulin  phosphatase (Dephosphorylation): protein that are active when dephosphorylated are anabolic (ex. PFK-1, Glycogen Synthase) Glutagon  kinase (Phosphorylation): proteins that are active when phosphorylated are catabolic (ex. G-6-Pase, Glycogen Phosporylase) 1. Discuss when and how fats are mobilized 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 A in 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 mobilization b. Transported in the blood bound to albumin 2. Describe how free (unesterified) fatty acids are transported in the blood. Free Fatty Acids are transported in the blood bound to Albumin. If the FFAs were unbound they would disrupt Cell Plasma Membranes in the blood vessels 3. Identify tissues where fatty acids oxidation occurs - Liver: can synthesize ketone bodies - Muscle: solely used for energy 4. Describe  -oxidation and discuss how energy is generated from this pathway. 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 Translocase transport FA-CoA into mitochondrial lumen. o Carnitine is synthesized or provided by diet  Synthesis requires Vit C - C12 or less can pass through transporter Peroxisomes:

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Shortens FA’s 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 H2O  8Acetyl 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 discuss when and where they are made and utilized - 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

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 bears hibernate for months without getting dehydrated. How do camels make it through 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 muscle mass reducation in the heart