PASS Anabolic pathways.pdf

Chapter Five: Enzymes The Most Important Proteins in the Body Enzymes are the most important proteins in your body. Their main job is to help you create energy. If I were to ask you whether a reaction is possible, the answer is always, yes. Anything, as we know, is possible. But, if I asked whether...

Chapter Five: Enzymes The Most Important Proteins in the Body Enzymes are the most important proteins in your body. Their main job is to help you create energy. If I were to ask you whether a reaction is possible, the answer is always, yes. Anything, as we know, is possible. But, if I asked whether something was probable, the answer now changes to no. 90% of reactions in the body would probably not occur without enzymes to catalyze the reaction. Sure, it may be probable, but we may have to wait a thousand years for it to happen! Enzymes make reactions more probable and easier to occur. Figure 5.1 Enzyme Reaction Figure 5.1: Here is an example of a reaction. There is a substrate or multiple substrates on one side. Now we must climb this mountain, which represents the free energy of activation (the amount you must climb before you do the reaction). At the top of the mountain is the high energy intermediate. This means that the substrate has so much energy that if you do not stabilize him, he takes off and you cannot finish the reaction. If you stabilize him and he stands still, the reaction slides down to the other side of the hill and you get the products that you want. Notice that, at the end, the enzyme is still intact. 129 Enzymes An Enzyme… 1. Brings substrates together in space and time 2. Lowers the free energy of activation—less energy needed for reaction • Activation energy—energy needed for chemical reaction to proceed 3. Stabilizes the high energy intermediate • Is not consumed in the reaction • Enzymes make improbable reactions occur 4. Makes reactions go faster Figure 5.2 Enzyme Binding Sites • • Vmax is equal to efficacy (when referring to drugs) Potency and Km have an inverse relationship Every enzyme has at least 2 sites: 1. ACTIVE SITE • where the substrate binds • where competitive inhibition will occur 2. REGULATORY SITE • where the enzyme is sped up (allosteric activator) or slowed down (allosteric inhibitor) • where non-competitive inhibition will occur 130 Enzymes Competitive Inhibition Non-Competitive Inhibition Substrate and inhibitor have similar properties and therefore bind to the same site Substrate and inhibition DO NOT have similar properties The Substrate and Inhibitor both bind to the same ACTIVE site The inhibitor binds to site other than active called the Allosteric site. Causes enzyme to change confirmation; substrate therefore cannot bind ↑ Km=substrate concentration, affinity decreases No change in Km, affinity stays the same ↑ Km will overcome inhibitor ↑ Km will NOT overcome inhibitor V max stays the same, efficacy stays the same V max decreases, efficacy of the substrate decreases Km=1/Potency, as Km increases potency decreases. Vmax = efficacy, which stays the same Km stays the same, so there is no change in potency. Vmax decreases; efficacy decreases *Majority of drugs (90%) are competitive inhibitors. This way, if patient overdoses, the effect can be reversed. *These types of drugs are used in diseases that are far worse in prognosis that the side effects of the drug. Clinical Correlation Why do we prefer drugs that act by competitive inhibition? They are reversible. Therefore, the majority of drugs “block” something else. Clinical Correlation When would we be justified to use a noncompetitive inhibitor? When the disease process is deadly (the benefit of the drug outweighs the risks of the drug itself) 131 Enzymes Figure 5.3 Gibbs Free Energy Equation DELTA G -free energy of a reaction. We want it to be negative. That means, the reaction is favorable and spontaneous. If a reaction is positive, going to the right, then the reaction is not favorable. However, it will be favorable going in the opposite direction, which will be negative. A negative Delta G means that there is energy left over after the reaction is done. ENTHALPY is heat, delta h. ENDOTHERMIC means the reaction is taking in heat. EXOTHERMIC means that heat is given off (exergonic). Delta H minus temperature (ΔH-T): this is how temperature can play a role in a reaction. Temperature will make a reaction more favorable because as it rises, the negative number increases. Delta S is ENTROPY, a positive change in the degree of randomness. This means the substrates must become more stable. Remember that Delta G is additive. The most stable bonds in the body are carbon-carbon bonds Ex: fat 132 Enzymes TEMPERATURE • • • • Can make a reaction more favorable Generates energy Can raise the BMR More likely to break up ATP and give off energy See Figure 5.4: • • • As temperature rises, Vmax increases At a certain temperature, Vmax will drop off quickly because Enzymes will denture. • • At 42 degrees C (106-107 degrees F), brain will denature Vaporized cool water: the best and most effective way to cool off the body, will decrease the risk of heat stroke REDOX POTENTIAL: • • • You want it to be negative When negative: has electrons to give away When positive: wants to accept electrons REMEMBER When you give away electrons, you get OXIDIZED When you accept electrons, you get REDUCED • • • REDUCING AGENT Has a negative E Wants to give away electrons Gets oxidized after the reaction • • • 133 OXIDIZING AGENT Has a positive E Wants to accept electrons Gets reduced after the reaction Enzymes Figure 5.4 General Redox Reaction Electron Transport Chain Found in the inner mitochondrial membrane 134 Figure 5.5 Electron Transport Chain Enzymes 135 Enzymes Every Complex in the Electron Transport System is positively charged, being more positive as you go down the chain • • • • • • • • Made up of four enzyme complexes and two electron carriers Complex I, II and IV—span the inner membrane • This allows them to pump protons from the inner mitochondria through the lipid bilayer of the inner membrane to the inner membranous space Inner membrane is where proton gradient is created Both Complex III and IV use heme Where we make 90% of ATP on any given day Copper is part of Complex IV • Also called cytochrome oxidase (where oxygen is used) Complex V is where phosphorylation occurs Two electron carriers float within the lipid bilayer of the inner membrane: • Cytochrome C • Coenzyme Q • Destroyed by Statins • Also called ubiquinone ELECTRON TANSPORT CHAIN—HOW IT WORKS……. Chemiosmotic theory: • • • • • • • • • As electrons move from complex 1,2, 3 and 4, the protons that separate from them are pumped into this space The mitochondria have a double wall separating cytoplasm from matrix ETC is located on the wall facing the matrix (in the inner mitochondrial wall) The osmolarity of that space will increase, causing protons to flow back into the matrix be. Cause cytoplasmic membrane is impermeable to protons. They have only one way to flow, back into the matrix. This return to the matrix can only occur at complex 5. As they cross the matrix membrane, we get their energy from them by regulating how fast they cross by concentration gradient. NADH, who drops off electrons at complex 1 has 3 chances to obtain energy. Therefore, NADH is worth 3 ATPs (actually 2.5) FADH2, which drops off electrons at Complex 2, has 2 chances to obtain energy Therefore, FADH2 is worth 2 ATPs (actually 1.5) 136 Enzymes • When NADH is created in the cytosol during glycolysis, it cannot cross the mitochondrial membrane and must use a shuttle to get to the ETC Complex I • • NADH goes to Complex I in the mitochondrial membrane, also called NADH dehydrogenase Complex I regenerates NAD+ The H+ is pumped by Complex I into the intermembranous space Coenzyme Q • • • The electron goes to the first electron carrier coenzyme Q, also called ubiquinone Coenzyme Q10, CoQ10 As the electron is passed to coenzyme Q, then transports it to Complex III Complex III • Coenzyme Q floats over to Complex III, then transfers the electron to it Complex III sends the H+ into the intermembranous space Cytochrome C • • Complex III next passes the electron to Cytochrome C At the same time, H+ is pulled in from the mitochondrial matrix Complex IV • • • • • Cytochrome C moves over to Complex IV and gives it the electron Complex IV is also called cytochrome oxidase complex Includes heme and copper Complex IV is responsible for attaching four electrons to an O2 molecule Once 4 electrons are passed to oxygen, 4 H+ are sent to the intermembranous space. The O2 is released from Complex IV as 2 H2O. Final tally of H+ sent in the proton gradient: • • Two electrons are taken from NADH and as those are passed down the ETC — H+ are sent into the intermembranous space at Complexes I, III and IV Two electrons are taken from FADH2 and as those e- are passed down the ETC — H+ are sent into the intermembranous space at Complexes III and IV 137 Enzymes Therefore, NADH is worth 3ATPs (actually 2.5) and FADH2 is worth 2 ATP (actually 1.5) FADH2 • • • FADH2 is formed in the mitochondria during the beta-oxidation of fatty acids by acyl-CoA dehydrogenase and in the citric acid cycle by succinate dehydrogenase, where it changes succinate to fumarate Succinate dehydrogenase is bound to the inner mitochondrial membrane There it does double duty, participating as both an enzyme of the citric acid cycle and the ETC, where it is called Complex II Complex II • • • • • • No H+ are pumped across at Complex II As it changes succinate to fumarate, succinate dehydrogenase produces FADH2 from FAD+ Then succinate dehydrogenase immediately switches jobs and becomes part of ETC as Complex II The 2 H+ are sent back into the mitochondrial matrix This means they cannot participate in the proton gradient and thus the making of ATP, the 2 e- are sent to coenzyme Q The reminder of the ETC for the e- that came off FADH2 is the same as that for e- from NADH Inhibitors and Uncouplers Inhibitors block electron transport and ATP synthesis • • • • • Complex 1: Amytal, Rotenone Complex 2: Malonate Complex 3: Antimycin Complex 4: CO (see below), CN, Chloramphenicol (see below) Complex 5: Oligomycin Carbon Monoxide Poisoning • • • • • CO binds iron 200 times stronger than oxygen Competitive inhibition of oxygen, Hb-saturation will be normal pO2 will be normal = dissolved oxygen; eventually decreases Treat with supplemental oxygen (increase substrate; ↑Km) The history is the best clue! 138 Enzymes PHARMCOLOGY: CHLORAMPHENICOL • • • • • • • Blocks the 50s subunit by blocking peptidyl transferase Covers all gram positives, including Staphylococcus aureus Covers simple gram negatives Covers Rickettsia Affects all rapidly dividing cells Aplastic anemia—dose-dependent and dose-independent Gray baby syndrome Clinical Correlation 3 things to know about antibiotics: • • • How they work What they cover Major side effects UNCOUPLERS • • • • • • Grab the protons in the space and drag them back into the matrix Causes loss of gradient Allows electron transport to continue but ATP synthesis stops Energy released as heat Body temp rises Muscles unable to relax • Malignant hyperthermia • Neuroleptic malignant syndrome 1. Dinitrophenol (DNP) 2. Aspirin • Reye’s syndrome 3. Free fatty acids • American diet • Brown fat Clinical Correlation Microsteatosis 1. Pregnancy 2. Tylenol poisoning 3. Reye’s Syndrome Macrosteatosis 1. Obesity 2. Alcohol 139 Enzymes HOW TO NAME ENZYMES (90%) • • First name of an enzyme is the name of the substrate Last name of an enzyme is what you did to the substrate Last names of enzymes: • Kinase • Carboxylase • Phosphorylase • Synthase • Isomerase • Synthetase • Epimerase • Dehydrogenase • Mutase • Hydrolase • Transferase • Thio- • Lyase Muscles need energy (ATP) to release a contraction ENZYMES and FUNCTIONS: 1. Kinase • Phosphorylates the substrate using ATP • Uses magnesium as a cofactor 2. Phosphorylase • Phosphorylates the substrate using free phosphate (Pi) 3. Isomerase • Creates an isomer from the substrate • An isomer has the same chemical make-up but a different structure 4. Epimerase • Creates an epimer from the substrate • An epimer has the same chemical makeup and structure but differ around one chiral carbon 5. Mutase • Moves a side chain from one carbon to another carbon 6. Transferase • Transfers a side chain from one substrate to another 140 Enzymes 7. Transaminases • Involved in transferring amino groups from or onto amino acids 8. Lyase • Cuts carbon-carbon bonds • Must use ATP 9. Carboxylase • Use carbon dioxide to create a carbon-carbon bond • Must use ATP • Uses biotin as a cofactor 10. Synthase • Multiple substrates are stacked together to create a product • No bonds are broken 11. Synthetase • Multiple substrates are stacked to create a product • No bonds are broken • However, ATP is used in the reaction. 12. Dehydrogenase • A cofactor is used in the reaction • Someone is going to lose a hydrogen 13. Hydrolase • Uses water to break a bond • Add –ase to which bond is being broken 14. Thio• Always added when a sulfur bond is broken in the reaction 141 Chapter Six: Anabolic Pathways Putting It All Back Together Chapter Sections: I. II. III. Organization of Anabolic Pathways Anabolic Pathways Cellular Cycle Organization of Anabolic Pathways The body breaks down sources of energy in the following order: 1. Plasma glucose 2. Liver glycogen 3. Proteins 4. Lipids 5. Ketones The body builds the sources back up in the same order they were broken down by anabolic pathways. Anabolic = Build State: well fed ANS control: parasympathetic active when enzymes are dephosphorylated Hormone: insulin Second messenger: tyrosine kinase (for insulin), parasympathetic cGMP Location: cytoplasm • • • As soon as you eat, you replenish your plasma glucose first After glucose returns to the liver, gluconeogenesis turns off Next, glycogen synthesis turns on o Occurs in 5 areas: o Skeletal muscle o Liver o Adrenal cortex 142 Anabolic Pathways o Intestinal wall o Cardiac o The liver and adrenal cortex are the only two organs that can mobilize glycogen, as glucose, into the bloodstream. • • • Plasma Glucose: Normal: <100 Pre-Diabetes: 100-126 Diabetes: >126 Drug that inhibits gluconeogenesis: Metformin, Phenformin Sulfonylureas Chlorpropamide SE: SIADH Tolbutamide Tolinase (tolazamide) Glimepiride Glyburide Glipizide Clinical Correlation Insulin • • • • • • Growth factor Rebuilds everything Excess causes excess growth Therefore, DM2 patients are obese. The more insulin they make, the more obese they become. The more obese they become, the more insulin they make. 143 Anabolic Pathways Pharmacology Sulfonylureas MOA: • Block the voltage-sensitive potassium channels • Keep potassium within the cell • Promotes insulin release Drugs: First Generation: • Chlorpropamide • Tolbutamide • Tolazamide Second Generation: Enhance peripheral glucose uptake • Glimepiride • Glyburide • Glipizide Side effects: • Hypoglycemia • Sulfa-associated side effects • Chlorpropamide: SIADH Thiazolidinediones MOA: • Bind insulin receptors and stimulate transcription Drugs: • Pioglitazone • Rosiglitazone • Repaglinide • Troglitazone Incretin Mimetics MOA: • Mimics incretins (glucagon like peptide-1 [GLP-1]) that are secreted by the intestinal wall in response to food • Incretins are deficient in NIDDM • Injected subcutaneously with breakfast and dinner GLP-1: • Potentiates glucose induced insulin release • Inhibits glucagon release • Inhibits GI secretion and motility • Inhibit appetite and food intake • Leads to weight loss Drugs: • Exenatide • Liraglutide • Dulaglutide • Semaglutide • Albiglutide Side Effects: • Cardiotoxic • Pancreatitis • MEN 2 Alpha Glucosidase Inhibitors MOA: • Block alpha-1-glucosidase • Inhibits glucose absorption (postprandial) Drugs: • Acarbose • Miglitol Biguanides MOA: • Inhibit gluconeogenesis • Causes weight loss • Decreases progression of pre-diabetes to diabetes Drugs: • Metformin/Phenformin Side effects: • Interacts with IV contrast to cause renal failure • If time permits, stop metformin for a few days, then perform study • If acute, stop metformin, give IV fluids, and add N-acetylcysteine to protect kidney • Metabolic acidosis Dipeptidyl Peptidase-4 Inhibitors MOA: • Inhibits degradation of endogenous GLP-1 • They are weight neutral Drugs: • Sitagliptin • Vildagliptin 144 Anabolic Pathways Amylin Analog MOA: • Decreases glucose absorption • Decreases appetite • Slows gastric emptying • Decreases glucagon secretion • Can be used by type 1 DM as well Drug: • Pramlintide • Aspart • Glulisine: action within 15 minutes Short Acting: • Regular Intermediate Acting: • NPH • Lente Long Acting: • Ultralente • Detemir: less daily serum insulin level variability than with NPH or glargine • Glargine Insulins Rapid Acting: • Lispro Anabolic Pathways Glycogen Synthesis Steps: 1. Begins with Glycogenin as the substrate. It has an OH group on Carbon 1 2. Glycogen contains a lot of carbon bonds. These bonds have no entropy therefore requiring UTP as a source of energy 3. UTP donates 1 phosphate to phosphorylate the OH group on glycogenin 4. UDP remains 5. UDP binds to the phosphate on the glycogenin 6. Glucose binds to the oxygen on the glycogenin 7. This process forms an alpha 1,4 bond 8. Steps 3-7 are repeated to continue forming alpha 1,4 bonds between glucose molecules REMEMBER Rate Limiting Enzyme: Glycogen Synthetase Phosphorylation results in too many electrons around each other. This creates entropy needed to push the pathway forward. Carrier for one sugar: UDP Dolichol: for multiple sugars 145 Anabolic Pathways Figure 6.1 Glycogen Synthesis, Steps 1-8 9. 10. 11. 12. Glycogen Synthetase can only add 8-10 glucose residues at a time After 8-10 residues, a Branching Enzyme will create a branch point. Branch points contain an alpha 1,6 bond. Every branch point contains an OH group sticking out which allows the glycogen to be soluble and easier to mobilize. After a branch point is made, Glycogen Synthetase will continue to make another chain of alpha 1,4 bonds. 146 Anabolic Pathways Figure 6.2 Glycogen Synthesis, Steps 9-12 REMEMBER Glycogen contains 2 types of linkages: Straight chain: Alpha 1,4 bond Branch point: Alpha 1,6 bond Glycogen Solubility: 1 gram of glycogen carries 3 grams water. Therefore, diet should be 40% carbohydrates. 147 Anabolic Pathways Clinical Correlation Anderson’s Disease: • • No branching enzyme Only straight chains Pentose Pathway Figure 6.3 Pentose Pathway 148 Anabolic Pathways Purpose of Pentose Pathway: • • To make end product: Ribose-5-Phosphate used in DNA and RNA synthesis To make byproduct: NADPH NADPHs are electron donors (reducing agents) Uses of NADPH: • • • DNA Synthesis Fatty Acid Synthesis RBC Repair (used by glutathione) Steps: 1. 2. 3. Glucose to Glucose-6-Phosphate (G6P) via hexokinase or glucokinase G6P to 6-phosphogluconate via G6P dehydrogenase (G6PD) Last Step: makes Ribose-5Phosphate This pathway also makes a 4-carbon and a 7carbon phosphorylated structure that the body cannot use. Two enzymes can convert these structures into a 3-carbon and 6-carbon structure that the body can use in glycolysis. PP Clue X-linked Recessive Enzyme Deficiencies: • G6PD • Fabry’s (alpha-galactosidase) • Hunter’s (iduronidase) • CGD (NADPH-oxidase) • Lesch-Nyhan (HGPRT) • Adrenoleukodystrophy (CAT-1) • Adenosine Deaminase • OTC • PRPP Synthetase • Tyrosine Kinase REMEMBER Rate Limiting Enzyme: Glucose-6-Phosphate Dehydrogenase (G6PD) Clinical Correlation G6PD: More common in Mediterraneans (protects them from malaria) 149 Anabolic Pathways Clinical Correlation Mcc of hemolytic crisis: 1. Infection 2. Drugs Management: Wernicke’sFigure Encephalopathy 1.3 Respiratory Epithelium Definitions Phosphorylated sugars cannot leave the cell and can pull in water, causing cells Reabsorption: Move things from the urine back into to swell and die. the blood Transketolase and transaldolase both use vitamins as cofactors. A deficiency in Secretion: Moving the blood outover to the either one vitamin will cause thethings otherfrom enzyme to take its function. However, urine for excretion in the kidney this cannot occur in the Wernicke’s area of the brain (posterior temporal lobe). Wernicke’s area only uses transketolase. An alcoholic, who is deficient in thiamine will not have his transketolase functioning properly. Therefore, if given glucose, he will enter the pentose pathway and those cells will build up 4 and 7-carbons sugars, which pulls water into cells, causing cells to swell and burst. This causes Wernicke’s encephalopathy and Wernicke’s Aphasia. Therefore, we administer 100 mg thiamine before we administer glucose. 150 Figure 6.4 Transketolase and Transaldolase Anabolic Pathways 151 Anabolic Pathways Amino Acid Synthesis Figure 6.5 Amino Acid Synthesis 152 Anabolic Pathways This pathway involves 20 transaminases: one for each amino acid. Transaminases take carbons from the Krebs Cycle and convert them into amino acids. 6 places where amino acids are made from carbon structures in the Krebs Cycle: • • • • • • Pyruvate o Alanine, serine, glycine Acetyl CoA o Lysine, leucine, phenylalanine, isoleucine, threonine, tryptophan Alpha ketoglutarate o Glutamate, glutamine Succinyl CoA o Phenylalanine, tryptophan, tyrosine Fumarate o Proline Oxaloacetate o Aspartate, asparagine 3 of these transaminases do the most work: • • • Alanine Transaminase (ALT) Aspartate Transaminase (AST) Gamma-Glutamyl Transaminase (GGT) GGT: • • Located only in the liver mitochondria. When an amino acid is breaking down: o AA + alpha-ketoglutarate → glutamic acid + carbons This reaction is reversible to make amino acids GGT Carbons + glutamic acid → AA + alpha ketoglutarate AST Oxaloacetate + Amino Acid→ Aspartate + Carbons ALT: Pyruvate + Amino Acid → Alanine + Carbons 153 Anabolic Pathways REMEMBER Location of Transaminases • • Alcohol destroys all membranes. Mitochondria GGT o (90%) o AST (10%) Cytoplasm o AST (50%) o ALT (50%) If the cell membrane is destroyed: • • • One AST and one ALT will leak out into the bloodstream Elevated AST, ALT 1:1 ratio This ratio is seen in: o Liver disease (hepatitis) o Muscle disease o Trauma o Infection If the mitochondrial membrane is destroyed: • • • • • • One AST and one GGT will leak out into the bloodstream In addition to the one AST and ALT leaked from the cell membrane damage → o Elevated AST, ALT 2:1 ratio o Elevated GGT This ratio is seen in: o Alcoholic hepatitis o Only alcohol can destroy the mitochondrial membrane Hepatic necrosis: o AST and ALT level in the thousands Fulminant hepatitis: o Hepatic encephalopathy o Evidence of GABA Cirrhosis: o Liver cannot do its most basic function o Albumin < 2 or Factor 7 low (PT Elevated) The amino acid synthesis process builds up a lot of NADH, GTP, and FADH2 for energy. This will lead to the inhibition of the Krebs Cycle by: Isocitrate Dehydrogenase and Pyruvate Dehydrogenase shutting down → 154 Anabolic Pathways Isocitrate will back-up → Citrate increases → Citrate is shuttled into cytoplasm for Fatty Acid Synthesis Fatty Acid Synthesis Steps: 1. 2. 3. 4. Figure 6.6 Fatty Acid Synthesis Citrate inhibits PFK-1 (glycolysis) and activates Acetyl CoA Carboxylase Citrate gets cleaved by a lyase into Acetyl CoA and Oxaloacetate (OAA). This requires ATP Acetyl CoA is carboxylated to make Malonyl CoA. This also requires ATP Citrate Shuttle: OAA gets converted into Pyruvate (produces an NADPH for FA Synthesis) → Pyruvate becomes OAA via Pyruvate Carboxylase (anapleurotic function) → OAA + Acetyl CoA = Citrate REMEMBER Rate Limiting Enzyme: Acetyl CoA Carboxylase 155 Anabolic Pathways Figure 6.7 Citrate Shuttle Figure 6.8 Fatty Acid 156 Anabolic Pathways Steps: (continued) Round 1 Steps: 5. 6. 7. 8. Odd number attacks an even number: Add Malonyl CoA (3Cs) to Acetyl CoA (2Cs) Decarboxylate • Gets rid of CO2 which drives the reaction forward Add a hydrogen from NADPH onto the oxygen of the inside carbon. Add another hydrogen from another NADPH molecule Steps 7 and 8 result in the formation of a water byproduct Round 1 produces four carbons. Each subsequent round adds two carbons each. To make a 16-carbon fatty acid, 7 rounds will occur. Palmitic acid: • The main fatty acid that the body makes daily For Fatty Acid Synthesis, the body: • • • Cannot synthesize beyond 16 carbons Can only create double bonds at least 3 carbons apart Cannot create any double bonds after C-10 REMEMBER How many rounds to make or break a fatty acid? (#Carbons /2) —1 How many NADPHs to make it? C-2 How many ATPs to make it? C-1 Saturated FA: no double bonds Unsaturated FA: has double bonds Omega FA: counting carbons from the right side Omega-3 FA: lowers serum cholesterol Omega-6 FA: elevate cholesterol Policosanol: Omega-3 Essential means: • The body cannot synthesize it • It can only come from the diet 157 Anabolic Pathways Essential Fatty Acids: • • Linolenic Acid Linoleic Acid (used to make Arachidonic Acid) Arachidonic Acid Pathway Figure 6.9 Arachidonic Acid Pathway Arachidonic Acid can enter either into the cyclo-oxygenase (COX) or lipo-oxygenase (LOX) pathway. The COX pathway synthesizes prostaglandins, and the LOX pathway synthesizes leukotrienes. Blockage of either one pathway forces the substrate into the other pathway. 158 Anabolic Pathways Types of Prostaglandins: Prostaglandin A-2: • • • Also known as: Thromboxane made by platelets Vasoconstriction Promotes platelet aggregation Prostaglandin E-1: • • • • • Vasodilator (w/ some vasoconstriction) Renal: dilates the afferent arteriole Cardiac: keeps the PDA open GI: increases mucus production for protection Specific PGE-1s: o Misoprostol  Treatment of aspirin or NSAID induced ulcers  Abortifacient (pregnancy test required) o Alprostadil:  pure vasodilator  Keeps PDA open Prostaglandin E-2: • Dinoprostone o Used for labor induction (dilates the cervix) Prostaglandin I-2: o o o o o Also known as: Prostacyclin Made by all endothelial cells Vasodilator (also dilates pulmonary vessels) Inhibits platelet aggregation Made primarily by COX-2 Prostaglandin F-2: • • • • • Vasoconstriction GU: responsible for dysmenorrhea Separates placenta after fetal delivery Abundant in semen Specific PGF-2s: o Carboprost • Abortifacient o Latanoprost 159 Anabolic Pathways • • Glaucoma Stimulates eyelash growth REMEMBER Location of COX Types: COX 1: GI tract COX 1 and 2: Joints COX 2: Vascular endothelium Leukotrienes: o o o o Types: LTC4, LTD4, LTE4 Also known as: SRS-A (slow reacting substance of anaphylaxis) Made by mast cells 4 to 8 hours after allergic response (late symptoms) The most potent bronchoconstrictor & vasoconstrictor REMEMBER SRS-A is broken down by aryl sulfatase o LTB-4 (and IL-8): Strong chemoattractant for neutrophils Clinical Correlation Aspirin-Sensitive Asthma Aspirin and NSAIDS block the COX pathway. This can result in shunting to the LOX pathway increasing SRS-A synthesis. This causes severe bronchoconstriction. Therefore, some patients can have asthma symptoms exacerbated by aspirin. Clue: nasal polyps 160 Anabolic Pathways Irreversible Cyclo-oxygenase Inhibitor: • Aspirin o Indications:  Anti-inflammatory  Analgesic  Anti-platelet  Anti-pyretic o Side effects: GI upset, bleeding, gastritis, thrombocytopenia, decreased platelet function, Reye syndrome, cinchonism  Cinchonism: Thrombocytopenia Hearing loss (CN 8) Tinnitus Clinical Correlation Aspirin is not indicated during viral infections for pediatric population because of risk of Reye syndrome Event RR pCO2 HCO3- pH Acid-Base Less than 20 minutes Stimulates resp. High rate Low No change High Respiratory Alkalosis 30-60 minutes Acid dissociates High Low Low Normal Combined Respiratory Alkalosis and Metabolic Acidosis >60 minutes GABA increases Low High Low Low Mixed Acidosis Reversible Cyclo-Oxygenase Inhibitors: • • NSAIDS Side effects: same as aspirin, except for no cinchonism and no Reye syndrome o Indomethacin  Most potent  Treats gout (renal failure or bone marrow problems) 161 Anabolic Pathways • • • • • • • •  Closes PDA Phenylbutazone o Second in potency Ibuprofen o MC over the counter Naproxen o Best for dysmenorrhea o High sodium load Baclofen o GABA effect o For back spasms Ketorolac o Morphine strength analgesic effect Diclofenac o Topical Ketoprofen o Topical Sulindac: contains sulfur REMEMBER Steroids are the most used, and most potent, anti-inflammatory drugs in America Actions of Steroids: Anti-inflammatory Actions: • • • • • Kills T-cells and eosinophils o Decreases immune system Inhibit PLP-A o Cannot make arachidonic acid Inhibits macrophage migration o So, cannot process antigens and allergens Stabilizes mast cells o So, cannot degranulate and release chemicals Stabilizes endothelium o Macrophages cannot get into tissues to process antigens 162 Anabolic Pathways o Prevents fluids from leaking out of vessels Physiologic actions: • • Proteolysis o Proteins break down Gluconeogenesis o Causes diabetes, weight gain, insulin resistance, etc. Steroids: • • • • • • • • • • • • • Prednisone o Main oral form Methylprednisolone o Main IV form Triamcinolone o Main inhaled form Budesonide o For pediatrics has Ciclesonide o For pediatrics Beclomethasone o Induce surfactant in fetus Betamethasone o Induce surfactant in fetus Hydrocortisone o Topical, injectable o Best drug to take the place of cortisol in adrenal insufficiency Dexamethasone o Crosses membranes fastest o DOC to induce surfactant Fludrocortisone o Takes place of aldosterone in adrenal insufficiency Cyproterone o Blocks DHT receptors in prostate cancer Megestrol o Increases appetite in cancer patients Fluticasone o Nasal spray 163 Anabolic Pathways • • Mometasone o Nasal spray Danazol o To treat endometriosis o Androgenic Mast cell stabilizers • • • Cromolyn Nedocromil Side effect: GI upset Management: Asthma 5 Steps Step 1: Mild, intermittent symptoms (every now and then): B2 agonist, PRN Step 2: “1:2 Rule:” if use is more than once a day or more than 2 flare-ups per week, then add: Under age 5: add cromolyn or nedocromil daily Over age 5: add an inhaled steroid daily Patient should also carry B2 agonist to use PRN Step 3: If uncontrolled, add a long-acting B2 agonist and steroid combination (AKA: LABAs) Patient should still carry B2 agonist to use PRN Step 4: If uncontrolled, add oral steroids daily; start with large dose for 2 weeks and then taper slowly Step 5: Omalizumab Leukotriene Receptor Blockers (LRBs) • • • Used with B2 agonists (adjunctive) Zafirlukast Montelukast Lipoxygenase Inhibitor: • Zileuton 164 Anabolic Pathways Triglyceride Synthesis Figure 6.10 Triglyceride Synthesis After fatty acids have been synthesized, they can be combined and stored as triglycerides. Glycerol-3-Phosphate is the backbone for complex triglycerides. This backbone comes from DHAP in glycolysis. Steps: A 16-carbon fully saturated fatty acid is attached to the first carbon (C1) of G3P.  It is now called: Lysophosphatidic Acid 1. A 16-carbon unsaturated fatty acid is attached to C2 of G3P. 2. It is now called: Phosphatidic Acid  Phosphatidic Acid is the backbone for complex phospholipids. It is used primarily by neuronal tissue. 3. CDP is a carrier for complex lipids. The following attachments onto C3 of phosphatidic acid 165 Anabolic Pathways 4. 5. 6. 7. 8. CDP-Choline → Phosphatidylcholine (Lecithin and makes surfactant) CDP-Ethanolamine → Found in nerve tissue Cardiolipin:  Combination of two Phosphatidic Acids Phosphatidylserine  CDP serine is added to phosphatidic acid  Cellular marker for apoptosis A 16-carbon fatty acid is attached to C3 of G3P is called triglycerides. Glycerol-3-Phosphate: The liver has the enzyme glycerol kinase so it can synthesize its own Glycerol-3-Phosphate. If an OH group is added to C3 in place of a phosphate, it makes DAG used in the IP3DAG second messenger system. Triglyceride Transport 3 transporters: • • • Chylomicrons o Made in GI tract o Transport triglycerides to endothelium (75%) and liver (25%) VLDL o Made in liver o 95% triglycerides, 5% cholesterol o Transport triglycerides to adipose for storage IDL o Transport triglycerides from adipose to everywhere else Figure 6.11 Triglyceride Transport 166 Anabolic Pathways Signs of High Triglycerides • • Xanthelasma: fat pads on eyelids, shoulder Pancreatitis: fatty infiltration of pancreas causes fat necrosis, inflammation Pharmacology: • • • • Inhibition of Triglyceride Absorption: o Orlistat o Blocks pancreatic lipase o SE: steatorrhea Ezetimibe o Block lacteals Enhance Lipoprotein Lipase Activity: o Gemfibrozil o Clofibrate  Associated with colon cancer (Off-market) Fenofibrate Block VLDL Production in Liver: • • Niacin o Side Effects: • Flushing and itching due to prostaglandin release, which stimulates mast cells to release histamine • Blocks insulin receptors • Competes with uric acid excretion to cause gout • Benefit: best drug to raise HDL • By inhibiting prostaglandins, aspirin prevents flushing and itching from niacin Probucol 167 Anabolic Pathways Sphingolipids Synthesis Figure 6.12 Sphingolipid Synthesis Steps: 1. 2. 3. 4. 5. 6. The original structure to start with is a C16-SCoA. Serine is attached to C16-SCoA via its carrier, CDP. C16 attached to nitrogen of the serine. It is now called: Ceramide o Ceramide is the backbone for complex sphingolipids If UDP adds one sugar to ceramide, it is now called a cerebroside. If dolichol adds many sugars to ceramide, it is now called ganglioside. If a phosphorylcholine is attached to the ceramide, it is now called sphingomyelin. Sphingomyelin is used primarily by neuronal tissue. Once the body utilizes the complex lipids, cells endocytose them into a lysosome and lysosomal acid hydrolases will break them down. If the lysosomal enzyme is not present, the lysosome will stay fused to the sphingolipid and form inclusions, resulting in a lysosomal storage disease. Lysosomal Storage Diseases: • Gaucher: o Glucocerebrosidase o Ashkenazi Jews o Gargoyle o Macrophages look like crinkled paper o Erlenmeyer flask legs 168 Anabolic Pathways • • • • • • • • o Can wipe out the entire bone marrow Fabry’s: o Alpha-galactosidase X-linked recessive o Cataracts o Early renal failure Krabbe’s: o Beta galactocerebrosidase or galactosylceramidase o Globoid bodies (macrophages swollen with sugar) Tay Sachs: o Hexosaminidase A o Ashkenazi Jews o Cherry red macula Sandhoff’s: o Hexosaminidase A & B o Cherry red macula Niemann Pick: o Sphingomyelinase o Zebra bodies (irregular areas of myelin) o Cherry red macula o Hepatosplenomegaly Metachromatic Leukodystrophy: o Arylsulfatase deficiency o MS equivalent in child 5-10 years old Hurler’s: o Iduronidase o Gargoyle features o Cataracts Hunter’s: o Iduronidase sulfatase o X-linked recessive 169 Anabolic Pathways Cholesterol Synthesis Steps: 1. Acetyl CoA + Acetyl CoA → AcetoacetylCoA 2. Acetoacetyl CoA + Acetyl CoA → HMG CoA via enzyme HMG CoA Synthase 3. HMG CoA → → Cholesterol via HMG CoA Reductase HMG CoA Reductase: • Most active at 8:00pm. Therefore, late-night meals are associated with weight gain and atherosclerosis. • Allosteric Activator: HMG CoA • Allosteric Inhibitor: dietary cholesterol • American Indians have the highest concentration of this enzyme. Therefore, they have a higher incidence of gallstones. Figure 6.13 Cholesterol Synthesis Cholesterol Transporter: • • • 170 LDL: remnant of VLDL and IDL LDL Receptor uses B-100 to deposit the cholesterol into different tissues Clathrin pits are markers for LDL receptors AnabolicGallstones Pathways Management: Virchow’s Triangle: 3 factors that increase gallstone formation: 1. Increased cholesterol 2. Decreased bile salts 3. Decreased lecithin Types of Gallstones: Cholesterol stones: yellow or clear (80%) Bilirubin stones: Green or black (hemolytic anemia) Brown stones: Due to infection (Salmonella or E. Coli) Location of Gallstones: 90% lodge in cystic duct, 10% lodge in common bile duct 3 signs of CBD Gallstone: 1. Pancreatitis 2. Increased alkaline phosphatase 3. increased WBC count and fever Cholecystitis S/S: RUQ pain Pain after fatty meal Murphy’s Sign: seen on physical exam Gallstone Ileus: Complication of longstanding gallstone. Erodes through gallbladder wall, falls into duodenum, and gets lodged at the ileocecal valve. Pneumobilia: air introduced into the biliary tract from the fistula of duodenum Xanthomas: • Associated with hypercholesterolemia • Large fat pads seen one extensor tendons especially Achilles’s tendon and elbow 171 Anabolic Pathways Figure 6.14 Lipid Transport Management: Cholesterol Cholesterol screening begins at age 35 If family history, obese child: age 5 Total Cholesterol • • • • < 200: total cholesterol goal for general population 200-240: Diet and exercise (20-30 minutes, 5x per week), Treat: if male with more than one risk factor or female with two risk factors 240 or above: Treat everyone HDL: above 45 LDL: • • • • < 100: LDL goal for general population < 70: Diabetics 100-130: Diet and exercise, Treat: if 2 or more risk factors >130: Treat everyone 172 Anabolic Pathways Pharmacology: Statins: • • • Best drug to treat hypercholesterolemia MOA: blocks HMG CoA Reductase o Simvastatin: decreases mortality in diabetics even if cholesterol is normal o Atorvastatin: decreases mortality in males with strong family history of hypercholesterolemia o Pravastatin: excreted via kidneys o Valdestatin: off the market because of severe side effects o Cerivastatin: off the market because of severe side effects o Rosuvastatin  Side effects: • Destroys Co-Q in ETC • Myositis  Check CPK if complain of weakness • If CPK >10x normal, add Co-Q • If myoglobin elevated, think rhabdomyolysis o Hepatitis If liver enzymes increase to more than 3 times above normal, start Coenzyme Q. If no change, then stop the statins Urea Cycle • • • Neither anabolic nor catabolic Active all the time Sources of nitrogen: o Glutamate o Aspartate Urea: • • • Water-soluble 90% synthesized by liver 10% synthesized by collecting duct (kidney) o Used to measure efferent blood flow (equivalent to renal plasma flow, PAH clearance, BUN clearance, secretion) o Used by ADH in water reabsorption o BUN increased by protein breakdown (normal urine output) 173 Anabolic Pathways Steps: In mitochondria: 1. Ammonia + CO2 + 2ATPs → Carbamoyl Phosphate via CPS-1 2. This step requires N-Acetyglutamate (N-Acetylglutamic acid) as allosteric activator 3. Carbamoyl Phosphate + Ornithine → Citrulline via Ornithine Transcarbamoylase In cytoplasm: 1. 2. 3. 4. Citrulline + Aspartate → Arginosuccinate via Arginosuccinate Synthetase Arginosuccinate cleaved into L-Arginine and Fumarate via Arginosuccinate Lyase L-Arginine splits into Urea and Ornithine via L-Arginase Fumarate enters TCA Cycle REMEMBER Rate Limiting Enzyme: Carbamoyl Phosphate Synthetase 1 (CPS-1) Glutamate delivers ammonia for the urea cycle Allosteric activator: N-acetylglutamate (Acetyl CoA and excess glutamate) meet up in the mitochondria. Signals to speed up urea cycle to get rid of the ammonia or else it will increase GABA. Urea Cycle Defects: • • Enzyme deficiency early in the pathway: • High ammonia (in the urine or plasma) Enzyme deficiency late in the pathway: • Serum pH is high • Pyrimidines in the urine 174 Anabolic Pathways Figure 6.15 Urea Cycle 175 Anabolic Pathways Management: Liver Failure Liver Failure: Most common NASH, followed by alcohol Decrease dietary protein: 2gram protein diet (Vmax of CPS-1) Clean out the GI tract of bacteria that can produce urea (ureasepositive bacteria): neomycin. Lactulose to flush out the GI. Wash out ammonia out of GI tract, ultimately pulls ammonia out of the brain. Contraindicated: • Fat-soluble drugs • P450-dependent drugs • Benzodiazepines • Barbs • Drugs that enhance GABA • Alcohol Clinical Correlation Hepatorenal Syndrome Glutamate cannot enter urea cycle. Add another amine group and turn it to glutamine. The liver does this to send the glutamine to the kidney where glutaminase will break it down. But the kidney can only do 10%. Kidney cannot makeup. Ammonium levels over time will rise. GABA will increase. GABA effect. Nucleotides • • • Used for DNA & RNA Used for Energy Carriers: • UDP: one sugar • Dolichol: multiple sugars 176 Anabolic Pathways • • SAM: methyl donation in entire body except for nucleotides THF: methyl donation in nucleotides THF:  Made from Vitamin B9  Made indirectly from Vitamin B12  Needed for nucleotide synthesis 3 ways to separate B12 from B9 deficiency: 1. Timing: • Folate is gone in 24 hours • B12 acts like it is fat-soluble, it is stored in your liver for up to 12 months. Takes 3-4 years to become deficient. 2. Neuropathy 3. Methyl-Malonyl CoA Mutase uses B12, so excess methylmalonic acid in the urine • • B9 and B12 Deficiency: Megaloblastic anemia with hyper segmented neutrophils B12 deficiency results in Neuropathy Types of Nucleotides: Purines: • • Adenine Guanine Pyrimidines: • • • Cytidine Uracil: found in only RNA Thymidine: found only in DNA Bonds: • Adenine makes two bonds with Thymidine in DNA • Adenine makes two bonds with Uracil in RNA 177 Anabolic Pathways • Guanine makes three bonds with Cytidine • A and T have a looser bond • G and C have a tighter bond Euchromatin: • Loose DNA • More As and Ts • Areas of loose DNA will be fast dividing areas. Therefore, rapidly dividing cells will have more As and Ts (i.e., skin) • To begin replication or transcription, must start in a loose area of DNA (i.e., area with more As and Ts)  CAAT and TATA box Heterochromatin: • Tight DNA • More Gs and Cs • Located in areas with slower dividing cells (i.e., brain) UV Light Damage • • • • Skin is the organ that is most susceptible to UV light damage Thymidine is the most susceptible to UV light damage If a cell is damaged by UV light it will form thymidine dimers (T-T bonds) Cells have an enzyme called UV light endonuclease within the helix that finds T-T bonds and snips them out Diseases related to UV Light Ecthyma or Ichthyosis • Partial deficiency of UV light endonuclease • Inherited Autosomal Recessive • Dry, flaky, lizard like skin Xeroderma Pigmentosa • Complete deficiency of UV light endonuclease • The DNA mutations cannot be removed at all • More susceptible to skin cancers 178 Anabolic Pathways Ataxia Telangiectasia • Too many DNA breaks from free radicals • Unable to repair all of them Lynch Syndrome (HNPCC) • Problem with mismatch repair Methylation • • • • A better way to tighten DNA than Gs and Cs is methylation If DNA is methylated, it cannot replicate or transcribe To stop or hide bad genes (i.e., oncogenes), they are methylated Requires energy  Low energy states (i.e., old age, chronic diseases) → oncogenes cannot be methylated → increased risk for cancers or disease in general  Transmission Rate: • In low energy states, bad genes are not hidden therefore more likely to pass on to next generation • Next generation will present earlier in life and more severe because the offspring’s initial energy level is lower than the parent’s (Anticipation) Clinical Correlation The Blots: • Southern Blot = DNA • Northern Blot = RNA • Western Blot = Protein • Southwestern Blot = DNA going to protein (translation) • PCR = amplifies DNA or RNA so the strips can be counted (used for viral load in HIV) • ELIZA = detects antibodies against anything #1 Risk Factor of Cancer: Old age and low energy 179 Anabolic Pathways Purine Synthesis Steps: 1. Start with Ribose-5-Phosphate from the Pentose Pathway 2. R5P + ATP → PRPP via PRPP Synthetase • PRPP decides which path to continue with: De Novo or Salvage • PRPP is high = De Novo is turned on, higher Km • PRPP is low = Salvage is turned on, lower Km De Novo: requires a lot of energy to run 3. PRPP + Glutamine → 6-Phosphoribosylamine + pyrophosphate (PPi) • This step used 2 ATPs 4. 10 more steps occur using 4 more ATPs (total of 6 ATPs utilized so far) 5. 10 steps created IMP (Inosine Monophosphate) 6. Fork in the pathway because IMP can give rise to either AMP (Adenine) or GMP (Guanine) (Inosine is the precursor of either purine) Cross Regulation: • To make AMP (Adenine), GTP is required as energy to stimulate it • To make GMP (Guanine), ATP is required as energy to stimulate it. Normal feedback inhibition is not the rule here. 7. To go from IMP to ATP (Adenine) = 3 more ATPs used • ATPs + 6 ATPs utilized earlier = 9 ATPs utilized to make 1 ATP (Adenine) 8. To go from IMP to GTP (Guanine) = 4 more ATPs used • 4 ATPs + 6 ATPs utilized earlier = 10 ATPs utilized to make 1 GTP (Guanine) REMEMBER Rate Limiting Enzyme: PRPP Synthetase 180 Anabolic Pathways Figure 6.16 Purine De Novo Synthesis 181 Anabolic Pathways Salvage Pathway: requires less energy than De Novo to run The body can take bases (hypoxanthine, guanine, adenine) from dying cells and recycles them into purines Hypoxanthine————> IMP + Pyrophosphate via HGPRTase • • uses 2 ATPs compared to the 6 ATPs required in De Novo to reach IMP Guanine—–— > GMP + Pyrophosphate via HGPRTase • uses 2 ATPs Adenine——–> AMP + Pyrophosphate via APRTase • uses 2 ATPs The body likes to run Salvage Pathway most of the time because it costs the least amount of energy. Figure 6.17 Purine Salvage Pathway When is De Novo preferred? During periods of rapid growth when the body needs so many nucleotides that it cannot wait for cells to die for its energy needs 182 Anabolic Pathways 3 Periods of Growth: • 0-2 years of age o They are tired during these years because they need so much energy o They eat, and sleep all day o This is a critical period because their whole body is rapidly dividing, including the brain o Never put a child who is under 2 on a diet, their growth will stunt o Think terrible 2s when they have energy again • 4-7 years of age o This is also the period in which children grow a preferential taste for certain foods o Must be careful what is fed to them during this period o Child’s preference for certain foods is set by age 8 o Maximum number of adipocytes is fixed by age 8 • Puberty PP Clue Patients who suffer from Cancer run De Novo PP Clue PP Clue What pathway is the body running when it is not in periods of rapid growth? Answer = Salvage Pathway Rapidly dividing cell release a substance called K.I. 67 as a signal to other rapidly dividing cells to start their rapid cellular division 183 Anabolic Pathways Pathological Periods when De Novo is turned on: • • Cancer (rapidly dividing cells) o Will begin to lose weight because they will ultimately consume proteins to run De Novo Chronic inflammatory disease o I.e., Crohn’s, Whipple’s disease o Causes rapid cell turnover which also results in weight loss o TNF-Alpha: responsible for rapid cell turnover • If hypoxanthine, guanine, or adenine are not properly utilized, xanthine oxidase can turn them into uric acid. • During sustained periods of excessive cellular growth and death, uric acid levels will be high. • Uric acid gets deposited in joints that are used the most because they get the most amount of blood supply. This will result in gout. • Podagra is the deposition of uric acid (crystals) in the thumb or big toe. • Gout contains mono sodium urate crystals. For the crystals to come together you need three things: 3 Things needed for Membrane Movement: • • • ATP Calcium Microtubules *Calcium crystals that are seen in patients with Gout are associated with intense pain. Gout Treatment: • Acute Gout: • Most Effective Drug:  Colchicine  MOA: blocks microtubules and interferes with cellular division and inflammatory cell mobility • Drug of Choice:  Indomethacin • In the Presence of Renal Failure: (Colchicine and Indomethacin are acidic drugs) 184 Anabolic Pathways  Steroids (injected into the affected joints) Chronic or Recurrent Gout:   Allopurinol or Febuxostat o MOA: blocks xanthine oxidase Probenecid o MOA: promotes uric acid excretion Effects of Chemotherapy on Rapidly Dividing Cells Patient with cancer → on chemotherapy → causes rapid cellular death → high levels of hypoxanthines, guanine, and adenine released from dying cells → xanthine oxidase turns them into uric acid → hyperuricemia and gout → treat chemo patients with Allopurinol and fluids to prevent hyperuricemia HGPRT:   Partial HGPRT Deficiency o Present with gout o Manifests in children (autosomal recessive) Complete HGPRT Deficiency o Called Lesch-Nyhan Syndrome o Severe gout o Self-mutilation o Always running De Novo pathway o Bone marrow is wiped out because cells are always rapidly dividing o X-Linked Recessive Enzyme Deficiencies Pyrimidine Synthesis Steps: 1. 2. 3. 4. 5. NH4 + CO2 + 2 ATPs → Carbamoyl Phosphate via CPS-2 Several subsequent steps First ringed structure made: Orotic Acid (white crystal) Orotic Acid + PRPP → UMP UMP can either make TMP or CMP Constituents:      Glycine- not involved Aspartate 185 Glutamine CO2 THF Anabolic Pathways Figure 6.18 Pyrimidine Synthesis 186 Anabolic Pathways • Difference between Uracil and Thymidine: Thymidine contains a methyl group (from THF) • Difference between Uracil and Cytidine: Cytidine contains an amine group from glutamine • Hydroxyurea: Drug used in Sickle Cell patients to increase HbF levels • 5-Flourouracil (5-FU): Drug inhibits Thymidylate Synthetase Ribonucleotide Reductase • The Purine and Pyrimidine pathways only made RNA nucleotides so far • Ribonucleotide Reductase converts RNA to DNA; ribonucleotides to deoxyribonucleotides • Enzyme works with Thioredoxin and NADPH • Enzyme recognizes only dinucleotides (ADP, GDP, CDP, UDP) • Contains 4 active sites and 4 regulatory sites • Enzyme blocked by drug Hydroxyurea • When enough is synthesized, each deoxy trinucleotide can inhibit its own site. • When enough dATP is synthesized, it shuts down the entire enzyme o dATP is the allosteric inhibitor of Ribonucleotide Reductase o Therefore, dATP must be made last o dATP levels must be kept low until the other 3 deoxynucleotides are made o Adenosine Deaminase: converts dATP to inosine → inosine urinated out → keeps dATP levels low → Ribonucleotide Reductase can keep working Figure 6.19 Ribonucleotide Reductase 187 Anabolic Pathways SCID: • • • • Adenosine Deaminase Deficiency dATP levels increase → Ribonucleotide Reductase activity decreases → DNA synthesis decrease → rapidly dividing cells effected most -→most concerning: bone marrow Fatal disease: all affected children used to be dead by age 2 Treatment: bone marrow transplant → extended children’s lives up to age 10 (and going!) DNA Helix Types: A form B form Z form Right-handed Right-handed Left-handed 11 base pairs per turn 10 base pairs per turn 12 base pairs per turn Basic terminology: • • • • • Read or proofread 3→5 Everything else: 5→ 3 Complementary: As complementary to T, Gs complementary to C o i.e.: DNA is 15% As; How many Ts? (15%), How many Gs? (35%) Nascent strand: strand of the DNA that is template for replication Daughter strand: new strand that is being synthesized Histones: • • • • Help bind DNA helix and forms nucleosomes Rich in lysine and arginine (basic amino acids) Have a positive charge which attracts the negative charge of DNA (DNA is negative because of phosphates) Types: • H1: linker protein, not part of the nucleosome • 2 of each of remaining histones to form an octamer • H2a 188 Anabolic Pathways • • • H2b • H3 • H4 Anti-histone antibodies seen in drug induced lupus. Drugs that cause drug induced lupus: HIPPPE • Hydralazine • INH • Phenytoin • Procainamide • Penicillamine • Ethosuximide Cellular Cycle Figure 6.20 Cellular Cycle 189 Anabolic Pathways DNA Replication: DNA-A protein SSB proteins Primase (RNA Polymerase) DNA Polymerase 3 DNA Polymerase 1 DNA Ligase Topoisomerase 1 Topoisomerase 2 190 Anabolic Pathways Types of RNA: • • • • Ribosomal RNA (rRNA) Most abundant Synthesized in the nucleolus of the nucleus 2 Types: o Free Floating Ribosomes:  Synthesize proteins that stay in the cytoplasm o Fixed Ribosomes:  Fixed to the rough endoplasmic reticulum  Synthesize proteins that are packaged for secretion out of the cell or to other organelles • Messenger RNA (mRNA) o Most variable (different sizes and shapes) o Synthesized during transcription o DNA → mRNA o 3’ end contains a Poly A tail o 5’ end contains a Guanosine cap with attached methyl group • Small Nuclear Ribonuclear Proteins (SNRPs) o Smallest o Responsible for splicing of mRNA during posttranscriptional modification • Transfer RNA (tRNA) o Used in translation o Key shaped o Anti-codon: located at the tip of the key; responsible for finding appropriate amino acid o 3’ end contains CCA nucleotides: appropriate amino acid binds here and costs 2 GTPs 191 Anabolic Pathways Transcription: Making mRNA from DNA 192 Anabolic Pathways Translation: Turning mRNA into a protein 193 Anabolic Pathways Codons: • • Start codon: AUG Stop codons: o UAA o UAG o UGA Wobble Theory: o The 5’ end of a codon is the most important. Changing the 5’ end will change the amino acid it codes for o The 3’ end of a codon is variable. Changing the 3’ end usually will not change the amino acid it codes for Pharmacology: o Aminoglycosides: o work at 30s subunit o block initiation factor o Tetracyclines: o block the binding to 30s subunit o blocks transfer RNA o Chloramphenicol o works at 50s subunit o blocks peptidyl transferase o Macrolides o block translocase o Clindamycin o also blocks translocation o Lincomycin o is not used anymore but is related to Clindamycin Compare and Contrast: • • • All the studies were done on prokaryotes because you cannot mess with human genes initially Once it was done on eukaryotes, they noticed it was the same process but by different names. DNA Polymerase Alpha does what Primase does DNA Polymerase gamma does only Mitochondrial DNA 194 Anabolic Pathways • • DNA Polymerase Delta and Epsilon do what DNA Polymerase 3 does. Delta is the leading strand and Epsilon is the lagging strand DNA Polymerase Beta does what DNA Polymerase 1 does (Remove Primers) Replication Forks: • • Eukaryotes have multiple replication forks because there are miles of DNA to replicate Prokaryotes (viruses) have 1 fork Transcription: • • • • Monocistronic: o Eukaryotes are monocistronic o One messenger RNA translates into only one protein Prokaryotes are polycistronic Virus can make every protein they need from one messenger RNA They have multiple starts and stops on one strip of genetic material Translation: • • Eukaryotes use Methionine Prokaryotes are F-Methionine Mutations: • • Frame-Shift o Can occur for one of 4 reasons:  Delete 1base • Insert 1 base  Delete 2 base • Insert 2 base o Disease presents early in life because, once a frame shift occurs, everything after that is messed up o The protein is not functional Point Mutations o Change in one base at one point o Transition mutation:  Mutated letter is in the same family 195 Anabolic Pathways • •  Example: G becomes an A, C becomes a U o Transversion mutation  Mutated letter is in a different family  Example: G becomes a U, A becomes a C Silent mutation o Change in one base but still codes for same amino acid o Asymptomatic Missense mutation o Change in one base and now codes for a different amino acid o Example: Sickle cell Anemia REMEMBER What Amino acid is substituted in Sickle Cell anemia? Valine for Glutamate PP Clue Hemoglobin C disease substitutes lysine for glutamic acid at position 6 of the beta chain • Nonsense mutation o Change in one base and it becomes a Stop codon o Protein stops prematurely o Disease presents early in life 196