BIOL214 lecture notes part 2.pdf

Carbohydrate & Mitochondrial Disorders Types of carbohydrate metabolism disorders - Carbohydrate metabolism is affected by enzymes, cofactors, substrates, allosteric binding, and hormones - These can largely be affected by diet - But metabolism can be greatly impacted by autoimmune an...

Carbohydrate & Mitochondrial Disorders Types of carbohydrate metabolism disorders - Carbohydrate metabolism is affected by enzymes, cofactors, substrates, allosteric binding, and hormones - These can largely be affected by diet - But metabolism can be greatly impacted by autoimmune and genetic disruption associated with these effectors Autoimmune: Type I diabetes - Consequence of autoimmune destruction of the pancreatic insulin-producing β-cells Autoimmune: Type I diabetes 5 - Insulin deficiency impacts glucose uptake (and therefore metabolism of glucose) - Hyperglycemia symptoms are: - Polyuria (frequent urination) - Polydipsia (increased thirst) - Polyphagia (increased hunger) - Weight loss Effect of type I diabetes on metabolism 1.Pancreas fails to secrete insulin 2.Insulin receptor not activated 3.Failure to recruit GLUT4 transporters to membrane (in skeletal muscle, cardiac muscle and adipose tissue) 4.Glucose not taken up by cells 5.&6. Insufficient glycolysis 7.&8. Insufficient TCA cycle and oxidative phosphorylation Type I diabetes (for interest) Long term impacts of hyperglycemia - Cardiovascular disease - Nerve damage - Kidney damage - blindness Treated with exogenous insulin and close management of diet Genetic disorders of carbohydrate metabolism Abnormalities in an individual’s DNA (from a single base pair to entire chromosome rearrangement) can impact carbohydrate metabolism, leading to various health conditions - Referred to as inborn errors of metabolism - i.e. the body can’t convert food into energy and remove waste efficiently - Usually caused by defects in carbohydrate metabolism enzymes Examples - Galactosemia → can’t convert galactose → accumulation → liver damage - Glycogen storage diseases - Fructose intolerance → deficient aldolase → can’t break down fructose → High BG and liver damage Glycolysis in disease - Glycolytic mutations are relatively rare due to the importance of this pathway - Majority of mutations in enzymes = cells can’t respire = cell death Examples: - Pyruvate kinase deficiency - Phosphofructokinase deficiency - + many more Pyruvate kinase deficiency - Erythrocytes (RBCs) obtain all their ATP from glycolysis - They don’t have mitochondria - A deficiency in pyruvate kinase (PK) results in erythrocytes with decreased energy - The link to hemolysis is not well understood - Buildup of glycolytic intermediates can increase the level of 2,3-bisphosphoglycerate (2,3-BPG) → decreases affinity of hemoglobin for O2 → affects tissue oxygenation Phosphofructokinase-1 (PFK-1) & cancer - Increase glycolysis is a hallmark of cancers - As PFK-1 is important for regulating glycolysis, mutations can cause cancer - Cancer mutations alter enzymatic activity and allosteric regulation, e.g. - R48C mutant has reduced citrate inhibition - N426S mutant partly relieves ATP inhibition - Therefore, glycolysis proceeds despite “stop” signal - Some mutations might inhibit glycolysis and push forward the pentose phosphate pathway (week 5), producing nucleotides that can help the rapidly growing cells Mitochondrial diseases (don’t need to memorise image) Mitochondrial genetic defects can drastically affect cellular respiration - can also be nuclear genes coding for enzymes involved in oxphosph Tissues with a high energy demand are particularly vulnerable - brain, nerves, retina, skeleton, cardiac muscle 1:5000 people have a disease-causing mutation in a mitochondrial protein Mitochondrial Genome Mitochondrial genome contains 37 genes - 13 code for proteins of respiratory chain - Remainder code for rRNA and tRNA for protein synthesis There can be 5 genomes per mitochondria Most proteins in the mitochondria are made by nuclear genes, synthesised on cytoplasmic ribosomes, then imported into and assembled in the mitochondria Heteroplasmy Cell division results in some daughter cells containing more diseased mitochondria than others - results in different phenotypes in the same tissue This muscle biopsy show red ragged fibres (clumps of diseased mitochondria) that are common when deletions occur in some mitochondrial genes Mutations in protein coding genes - Complex 1: Leber’s Hereditary Optic Neuropathy (LHON): not a multisystem disorder, but a highly selective degeneration of the optic nerve - Cytochrome b (part of complex III): exercise intolerance, myalgia - Complex IV (cytochrome c oxidase, COX): Myoclonic Epilepsy with Ragged Red Fibers (MERRF) - Complex V (ATP synthase): neuropathy, ataxia, and retinitis pigmentosa (decreased ATP synthesis rate and decreased stability of the FOF1 ATPase) Leber’s Hereditary Optic Neuropathy (LHON) (abbreviation can be used in exam) - Degeneration of retinal ganglion cells and their neuronal axons that leads to loss of central vision (predominantly young adult males) - Transmitted maternally as it is primarily due to mutations in mtDNA - LHON is usually due to one of three pathogenic mitochondrial DNA point mutations in the ND1/4/6, subunit genes of complex I of the ETC MERRF syndrome (abbreviation can be used in exam) - Myoclonic Epilepsy with Ragged Red Fibres (MERRF) - Muscle twitches (myoclonus), weakness (myopathy), and progressive stiffness (spasticity) - Muscle cells have red ragged fibres - Mutations in mt tRNALys found in 80% of cases Type 2 Diabetes Mellitus (don’t need to know this diagram) Insulin export hinges on ATP concentration in β-cells - When ATP exceeds a threshold, an ATP-gated K+ channel closes, depolarising the membrane, releasing insulin Mutations in the mt tRNALys or tRNALeu genes compromise ATP production - no insulin released - A rare form of Type 2 diabetes mellitus results Fructose bisphosphatase deficiency Deficiency of fructose 1,6-bisphosphatase, one of the enzymes in gluconeogenesis - Caused by one or more mutations In FBP deficiency - There is not enough fructose bisphosphatase for gluconeogenesis to occur correctly = no/low glucose made - May cause hypoglycemia and lactic acidosis - Glycolysis will still work, as it does not use this enzyme Glycogen storage diseases - Rare defects in glycogen synthesis or breakdown - Enzymes affected may include - Glycogen synthase, glucose 6-phosphatase, branching enzyme, debranching enzyme, phoshoprylase kinase, GLUT2, and others - Type Ia (Von Gierke disease) - Most common type of glycogen storage disease - (> 90% of all cases) - Mutations in glucose 6-phosphatase → enlarged liver, kidney failure, hypoglycemia Altitude sickness ↑ Altitude is coupled with ↓ atmospheric pressure - For every breath inhaled there is less O2 available - Our bodies are forced to work harder to metabolise Respiration increases to get enough oxygen for energy production Overcompensation leads to blood vessel dilation and excess blood in the brain - Causes swelling (cerebral oedema): hallucinations, vomiting, imbalance, headaches Increased blood flow/pressure causes fluid leakage into the lungs, leading to difficulty breathing, gurgling sounds, coughing, exhaustion - The body compensates by ↑ heart rate and BP, forcing more fluid into the lungs - Victim “drowns” Heart attack: myocardial infarction - Complete interruption of blood supply to a portion of the heart - In the absence of O2 the cells must rely on glycolysis for ATP - Stores of glycogen and phosphocreatine are rapidly depleted - Phosphocreatine is a readily available source for replenishing ATP - ATP becomes too low to maintain membrane ion pumps - Swelling, decreased pH from lactic acid buildup, heart failure Summary - Type I diabetes results from an inability to produce insulin – autoimmune - Errors in glycolytic enzymes are rare, but found in e.g. PK, PFK-1 - Mitochondrial diseases result from mutation of proteins important in mitochondrial cellular respiration steps - Leber’s Hereditary Optic Neuropathy (LHON) - MERRF Syndrome - Type 2 Diabetes mellitus (rare form) - Gluconeogenesis and glycogen storage can be affected - Fructose bisphosphatase deficiency - Glycogen storage diseases (Von Gierke disease) - At high altitude, O2 deficiency can lead to increased heart rate, brain swelling, vessel dilation and death - Heart attack leads to O2 deficiency, rapid depletion of ATP and energy stores, and breakdown in osmotic balance in cells Pentose Phosphate Pathway Carboyhydrate metabolism Role of the Pentose Phosphate Pathway Acts as a metabolic shunt (detour) - Diversion from glycolysis to produce… NADPH: an electron donor (reducing agent) - Reductive biosynthesis of fatty acids and steroids - Repair of oxidative damage Ribose 5-phosphate: a biosynthetic precursor of nucleotides - Used in DNA and RNA synthesis - Used in synthesis of some coenzymes - 5 carbon sugar (a pentose!) Ribose: kind of a big deal - ATP - RNA - NAD, NADP We need ribose for ATP We need ribose for RNA Note: in DNA is it deoxyribose. Hence deoxyribose nucleic acid. We need ribose to make NAD & NADP Nicotinamide adenine dinucleotide = NAD NADPH vs NADH: revision Both dinucleotide molecules can act as electron donors: - they act as cofactors in many biochemical reactions NADPH is especially important for reactions involving reduction - anabolism of fatty acids & steroids - Reduction of glutathione The pentose phosphate pathway is the major source of NADPH - NADH (can be oxidised to NAD+) - NADPH (can be oxidised to NADP+) Pentose Phosphate Pathway Pentose Phosphate Pathway PPP: Oxidative Phase What is the NADPH used for? Cells directly exposed to O2 (eye lens, red blood cells) are safeguarded from harmful free radicals - NADPH creates a reducing environment! This allows glutathione (GSSG) to be reduced… Glutathione Glutathione is a tripeptide consisting of glutamate, cysteine and glycine Glutathione - How does reduced glutathione help? - It’s an antioxidant! - Anti = opposed to - Oxidant = something that causes oxidation Glutathione Generation of ribose-5-phosphate Generation of ribose-5-phosphate (don’t need to know chemical structures, need to know substrate names and enzymes) 1. G6P is oxidised (loses 2 H) by glucose-6- phosphate dehydrogenase, forming 6- phosphoglucono-δ-lactone (+1 NADPH + H+) 2. Hydrolysis of 6-phosphogluconoδ-lactone by lactonase forming 6-phospho-gluconate Generation of ribose-5-phosphate cont… 3. Decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase to generate ribulose5-phosphate (+1 NADPH + H+) 4. Ribulose-5-phosphate can be converted via phosphopentose isomerase to ribose-5-phosphate or enter the nonoxidative phase of PPP Generation of ribose-5-phosphate 1. G6P is oxidised (loses 2 H) by glucose-6-phosphate dehydrogenase, forming 6-phosphoglucono-δ-lactone (+1 NADPH + H+) 2. Hydrolysis of 6-phosphoglucono-δ-lactone by lactonase forming 6- phospho-gluconate 3. Decarboxylation of 6-phospho-gluconate by 6-phosphogluconate dehydrogenase to generate ribulose-5-phosphate (+1 NADPH + H+) 4. Ribulose-5-phosphate can be converted via phosphopentose isomerase to ribose-5-phosphate or enter the non-oxidative phase of PPP Non-oxidative phase regenerated G6P from R5P - Reversible interconversion of sugars - Used in tissues requiring more NADPH than R5P - E.g. liver - Converts 6x pentose (5C) phosphates to 5x hexose (6C) phosphates Non-oxidative phase regenerates G6P from R5P - Oxidative pathway - ribulose-5-phosphate can be isomerised to ribose-5-phosphate by phosphopentose isomerase - Non-oxidative pathway – epimerisation of ribulose-5-phosphate by ribulose-5-phosphate epimerase - Xylulose-5-phosphate is then used in subsequent reactions Non-oxidative phase regenerated G6P from R5P Transketolase – reaction (1) (need to know these enzymes below) Transketolase catalyses the transfer of a two-carbon group from a ketose donor (xylulose-5-phosphate) to an aldose acceptor (ribose-5-phosphate) (via enzyme-bound TPP) Transketolase Transketolase catalyses the transfer of a two-carbon group from a ketose donor (xylulose-5-phosphate) to an aldose acceptor (ribose-5-phosphate) (via enzyme-bound TPP) - Generates a G3P for glycolysis & sedoheptulose-7- phosphate Transaldolase - Transaldolase removes a 3C fragment from sedoheptoluse-7-phosphate and is condensed with a G3P (3C) - Makes fructose-6-phosphate (6C) and erythrose-4-phosphate (acts as a substrate for the second transketolase reaction) The return of Transketolase - Transketolase returns to catalyse the transfer of a two-carbon group from a ketose donor (another xylulose-5-phosphate) to a different aldolase acceptor (erythrose-4- phosphate) - Makes glyceraldehyde-3-phosphate (3C) for glycolysis and fructose-6-phosphate (6) (can go on to create G6P) NADPH regulates partitioning into glycolysis vs PPP NADPH accumulation inhibits G6P dehydrogenase, while NADP+ stimulates it Inhibition occurs when NADPH is formed faster than it can be used for biosynthesis & reduction of glutathione This regulation controls the PPP’s activity based on the cell’s NADPH needs G6P dehydrogenase deficiency Can be fatal in cases of high oxidative stress - Certain drugs, herbicides, and some foods Confers resistance to malaria due to high oxidative stress in red blood cells G6P dehydrogenase deficiency - Primaquine and compounds found in fava beans (divicine) lead to increased amounts of peroxides and other reactive oxygen species - G6PD deficiency impairs the ability of red blood cells to maintain their levels of reduced glutathione, and therefore their ability to deal with oxidative stress. Pythagoras - Greek philosopher (yes, THE Pythagoras) - Prohibited his followers from eating fava beans (falafel) - Possibly because of favism - If people with Glucose-6-phosphate dehydrogenase (G6PD) deficiency eat fava beans: - Erythrocytes lyse - Haemoglobin is released into the blood - Jaundice and kidney failure can result NADPH and Malaria - Malaria parasite enters red blood cells and produces free radicals - G6PD deficiency have less NADPH → less defence against free radicals - Increases oxidative stress for red blood cell and parasite leading to death before it can complete its lifecycle What happens in the erythrocytes Summary The Pentose Phosphate Pathway is a process by which cells can generate reducing power (NADPH) that is needed for: - The biosynthesis of various compounds (via ribose 5-phosphate) - Reduction of oxidised glutathione (via NADPH) The non-oxidative phase can convert pentose phosphates back to glucose 6-phosphate via… - Transketolase (x2 reactions) - Transaldolase A deficiency in glucose 6-phosphate dehydrogenase leads to little reduced glutathione, and increased susceptibility to oxidative damage, particularly in erythrocytes (red blood cells) - This can provide an advantage for innate protection against malaria - Chemicals like divicine and ant-malarials can cause free radicals that can’t be dealt with Nucleotides Part I Nucleotides have Important Biological Functions - Storage of genetic information: DNA and RNA - Chemical energy: ATP, UTP, GTP and CTP - Components of major coenzymes: Coenzyme A, nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) - Regulation of cell metabolism: e.g. AMP and phosphorylation - Intracellular signalling: e.g. Cyclic AMP (cAMP) and phosphorylation - Extracellular signalling: Adenosine and P1 receptors, and ATP (and other nucleotides) and P2 receptors Nucleotide Structure: Revision Major Purine and Pyrimidine Bases of Nucleic Acids : Revision Nucleotide Nomenclature: Revision Two Pathways Lead to Nucleotides De novo pathways: Begin with metabolic precursors - ribose 5-phosphate, amino acids, CO2 and NH3 - and is metabolically expensive - Bases are synthesized while attached to ribose - Pyrimidine ring is synthesised as orotate - Glutamine provides most amino groups - Glycine is the precursor for purines - Aspartate is the precursor for pyrimidines Salvage pathways: Recycles the free bases and nucleosides released from nucleic acid breakdown and is metabolically economical (next lecture) Origin of Ring Atoms in Purines De Novo Purine Synthesis Begins with PRPP (focus on first step and end product) - Purine ring assembled, atom by atom, on 5- phosphoribosyl 1-pyrophospate (PRPP) - In the first committed step, an amino group donated by glutamine is attached at C-1 of PRPP to form 5-phosphoribosylamine - In the second step, three atoms are added from glycine, requiring ATP - A further eight steps are required in higher eukaryotes to form the ring structure and the nucleotide inosinate (IMP) (nine steps in bacteria; see Figure 22-35 for all 10-11 steps) De Novo Purine Synthesis Results in the First Complete Purine Inosinate (IMP) - In the tenth step, a second ring closure occurs to form inosinate (IMP) - IMP is the first intermediate with a complete purine ring - In addition to its central role in purine synthesis, it is used as a flavour enhancer (E631) in a variety of foods Inosinate (IMP) Forms a Branch Point in De Novo Purine Synthesis - Conversion of IMP to AMP requires an amino group from aspartate, requiring GTP - Conversion of IMP to GMP requires an amino group from glutamine, requiring ATP (only need to know highlighted and enzymes) Purine Biosynthesis is Regulated by Feedback Inhibition Four major feedback mechanisms cooperate in regulating the: - Overall rate of de novo purine nucleotide synthesis - Relative rates of AMP and GMP synthesis (diagram is like as summary) Pyrimidine Nucleotides are Made from Aspartate, PRPP and Carbamoyl Phosphate - Pyrimidine synthesis proceeds by first making the pyrimidine ring (in the form of orotate) - Aspartate and carbamoyl phosphate provide the atoms for the ring structure, making synthesis pyrimidine ring simpler than purine ring - Ribose 5-phosphate is attached after pyrimidine ring formation Carbamoyl Phosphate Synthetase II - Cytosolic enzyme that makes the carbamoyl phosphate required in pyrimidine biosynthesis (know this enzyme) - Different from carbamoyl phosphate synthetase I in the mitochondria required in urea cycle (see Urea Cycle lecture later in Session) - Catalyses: 2 ATP + L-glutamine + HCO3 - → 2 ADP + Pi + L-glutamate + carbamoyl phosphate De Novo Pyrimidine Synthesis Ribonucleotides are the Precursors of Deoxyribonucleotides - Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides - Thioredoxin acts as an intermediate hydrogen-carrying protein that carries a pair of hydrogen atoms, via –SH groups, from NADPH to the ribonucleoside diphosphate - Thioredoxin reductase catalyses the reduction of the oxidised form of thioredoxin by NADPH Reduction of Ribonucleotides to Deoxyribonucleotides - Reaction catalysed by ribonucleotide reductase (know this enzyme) - Electrons and H+ are transmitted from NADPH to ribonucleotide reductase via glutaredoxin and thioredoxin - Glutaredoxin transfers electrons from glutathione (GSH) to ribonucleotide reductase - Thioredoxin transfers pair of H+ to ribonucleotide reductase required to form dNDP (don’t need to know the details, mainly electron flow) Structure of Ribonucleotide Reductase Activity and substrate specificity of ribonucleotide reductase is regulated by the binding of effector molecules (don’t need to know details, just understand process) Regulation of Ribonucleotide Reductase by Deoxynucleoside Triphosphates (don’t need to know details, just ATP and dATP roles in regulating overall production) Thymidylate (dTMP) is Derived from dCDP and dUMP - DNA contains thymine rather than uracil - De novo pathway to thymine only involves deoxyribonucleotides - dUTP formed by deamination of dCTP or by phosphorylation of dUDP - Thymidylate synthase catalyses the conversion of dUMP to thymidylate (dTMP) (focus on first and last part of diagram) Conversion of dUMP to dTMP is Catalyzed by Thymidylate Synthase - Thymidylate synthase catalyzes the conversion of dUMP to dTMP - Dihydrofolate reductase reduces dihydrofolate to tetrahydrofolate - Folic acid deficiency results in excess incorporation of uracil into DNA leading to DNA damage causing neural tube defects (see slide 28) (don’t need to know details) Folic Acid Deficiency Leads to Reduced Thymidylate Synthesis - Folic acid (or vitamin B9) deficiency occurs in ~10% of the human population (and up to 50% of people in impoverished communities) - Reduced thymidylate synthesis causes abnormal incorporation of uracil into DNA - Repair mechanisms remove the uracil by creating strand breaks that affect the structure and function of DNA - Neural tube defects in infants (during pregnancy) - Associated with cancer, heart disease, and neurological impairment Thymidylate Synthesis and Folate Metabolism as Chemotherapy and Antibiotic Targets (don’t need to know details) Summary: De Novo Purine Nucleotide Synthesis Summary: De Novo Pyrimidine Nucleotide Synthesis Nucleotides Part II Revision: Two Pathways Lead to Nucleotides De novo pathways: Begin with metabolic precursors - ribose 5-phosphate, amino acids, CO2 and NH3 - and is metabolically expensive - Bases are synthesized while attached to ribose - Purine ring is synthesised while attached to ribose - Pyrimidine ring is synthesised as orotate then attached to ribose - Glutamine provides most amino groups - Glycine is the precursor for purines - Aspartate is the precursor for pyrimidines Salvage pathways: Recycles the free bases and nucleosides released from nucleic acid breakdown and is metabolically economical (next lecture) Revision: De Novo Purine Nucleotide Synthesis Revision: De Novo Pyrimidine Nucleotide Synthesis (linear) Purine and Pyrimidine Bases are Recycled by Salvage Pathways De novo pathway (last lecture) - PRPP (activated ribose) + amino acids + ATP + (CO2) + … → Nucleotide Salvage pathway - PRPP (activated ribose) + base → Nucleotide Purine Bases are Recycled by Salvage Pathways - Free purines can be salvaged and rebuilt into nucleotides - Adenosine phosphoribosyl-transferase catalyzes the reaction of a free adenine with PRPP to yield the corresponding adenine nucleotide - Adenine + PRPP → AMP + PPi - Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the salvage of free hypoxanthine (the deamination product of adenine) and guanine to form IMP and GMP, respectively - Hypoxanthine + PRPP → IMP + PPi - Guanine + PRPP → GMP + PPi - Salvage pathways exists for pyrimidine bases in microorganisms and possibly in mammals - Thymidine phosphorylase catalyzes thymine (released from degraded DNA) to thymidine - Thymine + Deoxyribose-1-phosphate ⇌ Thymidine + Pi - Thymidine kinase catalyzes thymidine to TMP - Thymidine + ATP ⇌ TMP + ADP - The anti-viral acyclovir is converted into a suicide inhibitor by viral (but not host) thymidine kinase and then by host kinases to to acyclovir triphosphate to terminate viral DNA synthesis in herpes simplex infections Nucleoside Monophosphates are Converted to Nucleoside Triphosphates - Adenylate kinase phosphorylates - AMP to ADP ATP + AMP ⇌ 2 ADP - ADP is phosphorylated to ATP by glycolytic enzymes or through oxidative phosphorylation Nucleoside Monophosphate and Diphosphate Kinases - Nucleoside monophosphate kinases use ATP to form other nucleoside diphosphates - ATP + NMP ⇌ ADP + NDP - Nucleoside diphosphate kinases convert nucleoside diphosphates to triphosphates - NTPD + NDPA ⇌ NDPD + NTPA - where A is the phosphate acceptor and D is the donor Purine Degradation Produces Uric Acid In primates most urea excreted via urea cycle (see Nitrogen Metabolism lecture) ( don’t need to know details, focus on 5’-nucleotidase, adenosine deaminase, xanthine oxidase) Altered Purine Degradation Leads to Disease DNA and RNA: Revision Adenosine Deaminase Deficiency Results in Primary Immunodeficiencies - Adenosine deaminase (ADA) deficiency leads to an accumulation of deoxyadenosine leading to a 100-fold increase in dATP, a strong inhibitor of ribonucleotide reductase - Causes severe immunodeficiency disease (SCID) in which T and B lymphocytes do not develop properly - Treated by - Donor blood stem cell transplantation - Enzyme replacement therapy - Gene therapy Lesch-Nyhan Syndrome - Genetic disorder in boys caused by a lack of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity, which salvages hypoxanthine and guanine to form IMP and GMP, respectively - In the absence of HPRT, PRPP and purine synthesis increases leading to excess uric acid production and tissue damage especially the central nervous system - Characterized by poor coordination, intellectual deficits, hostility, and compulsive selfdestructive tendencies Excess Uric Acid Causes Gout - Gout is an inflammatory disease of the joints caused by an elevated concentration of uric acid and urate crystals in the blood and tissues (joints) - Often involves an underexcretion of urate - Genetic deficiency of one or another enzyme of purine metabolism may also be a factor - Allopurinol is an inhibitor of xanthine oxidase that is used in the treatment of gout - Oxypurinol remains tight bound to enzyme Pyrimidine Degradation Produces Urea, Acetyl-CoA and Succinyl-CoA - Pathways for degradation lead to NH4 + production and thus to urea synthesis - Carbons of cytosine and uracil are degraded to acetyl-CoA - Carbons of thymine are degraded to succinyl-CoA Nucleotides and Nucleosides Function as Extracellular Signalling Molecules - 1929: ATP discovered - 1929: Extracellular purines act on muscles - 1970: Extracellular ATP identified as a neurotransmitter - 1972: Purinergic signalling hypothesis proposed by the late Geoffrey Burnstock - 1976: Extracellular ATP proposed as a coneurotransmitter by Burnstock - 1978: P1 and P2 receptors classified (don’t need to know details) Purinergic Signalling Pathways ADA, adenosine deaminase; Ado, adenosine; 5ʹ-NT, ecto-5ʹ-nucleotidase (CD73); E-NTPDase, ectonucleoside triphosphate diphosphohydrolase; E-NPP, ecto-nucleotide pyrophosphatase/ phosphodiesterase; Ino, inosine Purinergic Signalling has Various Roles in Health and Disease - P2Y12 activation by extracellular ADP is central to platelet activation and blood clotting - P2Y12 drugs are widely used in coronary heart disease Purinergic Signalling has Roles Graft-versus-host Disease (GVHD) (don’t need to know details) Enzymes to Know from this Lecture - Kinases (in general) = catalyse exchange of phosphates between nucleotides - Adenylate kinase = converts AMP to ADP - 5’ Nucleotidase = converts AMP to adenosine - Adenosine deaminase = converts adenosine to inosine - Xanthine oxidase = converts hypoxanthine to xanthine and xanthine to uric acid - HPRT (hypoxanthine-guanine phosphoribosyltransferase) = salvages hypoxanthine and guanine to form IMP and GMP - Nucleoside triphosphate diphosphohydrolase = converts ATP to ADP and ADP to AMP - Ecto-enzyme = cell surface or extracellular enzyme Summary - Purine and pyrimidine nucleotides are made by both de novo and salvage pathways - Kinases catalyse the exchange of phosphates between nucleotides - Purines are degraded to uric acid, and pyrimidines are degraded to urea, acetyl-CoA and succinyl Co-A - Defects in nucleotide degradation can cause disease - Extracellular nucleotides and nucleosides can function as cell signalling molecules Dietary and Circulating Lipids Lipids Have Important Biological Functions 1. Triacylglycerols 2. Fatty acids 3. Phospholipids 4. Glycolipids 5. Lipid hormones 6. Pigments Lipids: Revision Steroid nucleus gives rise to what main sterol in animals? Don’t need to know how to draw the structures Common Types of Lipids Digestion of Dietary Lipids - Most dietary lipids are triacylglycerols (TGs) - TGs must be degraded by enzymes to monoacylglycerols and fatty acids to cross the intestinal epithelium - Digestive enzymes are water soluble - Lipids (unlike carbohydrates and proteins) are not water soluble Bile Salts Emulsify Dietary Lipids to Allow Their Digestion - Bile acids are made from cholesterol and conjugated to glycine or taurine to form bile salts in the liver - Bile salts (or conjugated bile acids) are released via the gall bladder into the small intestine - Bile salts are amphipathic molecules - Examples of bile salts include conjugated cholic acid and its derivative taurocholic acid - In the small intestine, bile salts emulsify dietary lipids into micelles (lipid droplets) making the triacylglycerols more accessible to digestive enzymes to aid their digestion Triacylglycerols are Digested to Monoacylglycerols and Fatty Acids By Lipases in the Intestine This pathway is the topic of the Lipid Metabolism Practical Lipases Degrade Triacylglycerols in Various Tissues of the Body Three main types lipases: - Intestinal (pancreatic) - Lipoprotein - Hormone-sensitive Digestion of Dietary Lipids Plasma Lipoproteins - Particles which carry triacylglycerols, cholesterol, cholesteryl esters, phospholipids and lipid-soluble vitamins - Composed of proteins called apolipoproteins with various lipids - Apolipoproteins: - Solubilise lipids - Act as signals to target lipoproteins to specific tissues - Act as signals to activate enzymes Human Plasma Lipoproteins - Multiple types which differ in density and composition - Chylomicrons: form in intestinal mucosa and travel through the lymph and blood to other tissues (see above slides) - Very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL): originate from liver and extrahepatic tissues (don’t need to know the numbers in the table) Lipoproteins and Lipid Transport (don’t need to know the details) Apolipoproteins of Human Plasma Lipoproteins (don’t need to know details) LDL (Cholesterol) Uptake is Mediated by Receptor-mediated Endocytosis LDL (Cholesterol) Uptake is Mediated by Receptor-mediated Endocytosis Familial Hypercholesterolemia - Genetic disorder discovered by Michael Brown and Joseph Goldstein - Absence or deficiency in LDL receptors - 4-fold higher plasma cholesterol and LDL concentrations - Excess blood LDL is oxidised and engulfed by macrophages to form foam cells and atherosclerotic plaques in blood vessels - Homozygotes (both gene copies mutated) die of coronary heart disease in childhood unless treated by liver transplant - Heterozygotes (one gene copy mutated) have a milder and variable clinical disease and treated using drugs (statins) that reduce blood cholesterol (via HMG-CoA reductase inhibition), which in turn increase LDL receptors Formation of Atherosclerotic Plaques Formation of Atherosclerotic Plaques Summary - Lipids have important roles in biology - Lipids comprise different structures and functions - Dietary lipids are degraded by intestinal lipases before being absorbed and transported throughout the body - Plasma lipoproteins are important in the transport of lipids throughout the body - Deficits in lipid transport and metabolism can cause cardiovascular disease Lipid and Fatty Acid Degradation Adipose Tissue White adipose tissue - Stores excess calories as triacylglycerols that can be used as fuel (via b oxidation) to produce large amounts of ATP (via respiratory chain, oxidative phosphorylation) Brown adipose tissue - Stores triacylglycerols that are used as fuel (via b oxidation) to maintain core body temperature in response to cold (thermogenesis) Triacylglycerols (Triacylglycerides) Energy stores housed as: - Fat droplets in adipocytes (fat cells) - Oils in plant seeds Main advantages as energy stores: - Fatty acid carbons more reduced than those of sugars, thus greater energy content per unit mass - Hydrophobic and stored unhydrated, thus reduced water load for host Triacylglycerols: Revision (don’t need to memorise the structure) Hydrocarbon tail also referred to as the acyl chain Lipases: Revision How does the pathway on the left differ to the degradation of dietary triacylglycerols (triacylglycerides) in the intestine? - Degradation of dietary triacylglycerols terminates at monoacylglycerols and fatty acids (previous lecture) - Degradation adipocyte triacylglycerols terminates with glycerol and fatty acids Metabolism: Revision Degradation of Triacylglycerols Degradation of Triacylglycerols Activation of hormone sensitive lipase (HSL) initiates degradation of triacylglycerols in lipid droplet including involvement of other lipases Degradation of Triacylglycerols Results in Glycerol and Fatty Acids Lipids: Revision Oxidation of Fatty Acids β oxidation: - Repetitive four-step process by which fatty acids are converted to acetyl CoA - Occurs at C-3 or β position - Removal of successive carbon pairs from fatty acids - Occurs within mitochondria Some fatty acids undergo: - α oxidation within peroxisomes - ω oxidation within endoplasmic reticulum - β oxidation occurs in the mitochondria. - Fatty acids of ≤12 carbons can enter mitochondria directly - Longer fatty acids (≥13 carbons) are first linked to coenzyme A. - These activated fatty acids are then linked to carnitine to form fatty acyl carnitine. - Fatty acyl carnitine enters via carnitine shuttle. - Fatty acids of ≥14 carbons comprise the majority of dietary or released fatty acids. Revision: Coenzyme A is an Activated Carrier Important in the Oxidation of Pyruvate and Fatty Acids (don’t need to know details and structures) Acetyl-Coenzyme A is a Metabolic Intermediate Derived from Coenzyme A During fatty acid metabolism the thio group (SH) of coenzyme A (CoA) is substituted with the fatty acid acyl chain to form fatty acyl-CoA (don’t need to know details and structures) Fatty Acids are Linked to Acyl-Coenzyme A (CoA) to Form Fatty Acyl-Co (don’t need to know details) Long Fatty Acids are Converted to Fatty Acyl-CoA and Enter into Mitochondria via the Carnitine Shuttle Stages of Fatty Acid Catabolism Stage 1: - Fatty acids undergo β oxidation - Removal of successive carbon pairs (starting from carboxyl end) - These 2 carbons form acetyl -CoA - Eg: palmitic acid (16:0) directly yields 8 acetyl -CoA molecules (and 7 FADH 2 and 7 NADH molecules) Stage 2: - Acetyl -CoA enters the citric acid cycle Stage 3: - Stages 1 and 2 result in the electron carriers FADH 2 and NADH - The electron carriers donate electrons within the respiratory chain complex to produce ATP (and H2O) Each Round of b Oxidation Includes Four Key Steps (just need to understand the starting point and end products and the steps involved, not details or structures) Each Round of b Oxidation Includes Four Key Steps Pathway to the left is showing the same pathway as the previous slide, but is an example how different texts present the same concepts differently β Oxidation of Fatty Acids Leads to ATP Production, as Well as CO2 and H2O Production (don’t need to know detail, know the highlighted parts in both and need to know the “math” including acetyl-CoA) Oxidation of Unsaturated and Odd-chain Fatty Acids Requires Additional Steps - β oxidation causes the complete degradation of saturated fatty acids with an even number of carbons. - What about unsaturated fatty acids and odd-chain fatty acids? - The oxidation of unsaturated fatty acids involves an isomerase and a reductase*. - Odd-chain fatty acids yield propionyl CoA which is converted to succinyl CoA, which enters the citric acid cycle. - *Isomerase moves double bond; reductase removes double bond β Oxidation of Odd-chain Fatty Acids (don’t need to know details and structures) Vitamin B12 Deficiency Promotes Disease - Results from reduced absorption by gut or reduced dietary intake - Impairs (see brown box): Mitochondrion. Methylmalonyl-CoA metabolism → spinal cord degeneration Cytosol. DNA synthesis → megaloblastic anaemia (don’t need to know, brown box for understanding) Gut Microbiota-derived Short Chain Fatty Acids (SCFAs) Have Local and Systemic Effects (e.g. Cardiovascular) Lipid and Fatty Acid Degradation: Summary Appendix Fatty Acid and Lipid Synthesis Common Types of Lipids Lipid and Fatty Acid Degradation: Revision Revision: Metabolism Fatty Acid and Lipid Synthesis Fatty Acids Synthesis - Fatty acid synthesis takes place in the cytoplasm. - But fatty acid synthesis requires acetyl-CoA produced in mitochondria and CoA cannot cross mitochondrial membranes. So how do acetyl-CoA or acetyl groups gain access to the cytoplasm? - Acetyl groups are transported across the mitochondrial membrane into the cytoplasm in the form of citrate. Acetyl-CoA is Transported as Citrate from the Mitochondria (don’t need to know the enzymes) Acetyl-CoA is Converted to Malonyl-CoA by Acetyl-CoA Carboxylase - Helpful tip: Carboxylases are enzymes which transfer a carboxyl group (CO2) to a substrate - Helpful tip: Synthases are enzymes that catalyse synthesis (regardless of requirement for nucleoside triphosphates, e.g. ATP) Synthetases are enzymes that use nucleoside triphosphates to catalyse synthesis (need to know these enzymes) Fatty Acid Synthesis Involves the Addition of Two Carbons to an Acetyl Molecule in a Four-step Process via the Fatty Acid Synthase - Acyl carrier protein (ACP) shuttles the acyl groups between active sites of fatty acid synthase (FAS) during fatty acid synthesis - ACP is derived from pantothenic acid (vitamin B5) - What other common molecule is derived from vitamin B5? Coenzyme A (CoA) Malonyl-CoA is Used to Form Palmitate by the Fatty Acid Synthase Palmitate Serves as a Precursor of Other Long-chain Fatty Acids - Palmitate is lengthened by the fatty acid elongation system on the endoplasmic reticulum membrane - Coenzyme A (rather than ACP) is the acyl carrier - Mammals lack the ability to introduce double bonds beyond carbon position 9 - Thus, mammals require essential omega fatty acids in their diet (don’t need to know details) Omega Fatty Acids Are Essential in Human Diets - Also known as polyunsaturated fatty acids (PUFAs) or n fatty acids - Essential omega 3 fatty acids are shown to the right - Omega 6:omega 3 ratio is important - Optimal dietary ratio = 1:1 to 4:1 - Western dietary ratio = 10:1 to 30:1 - Higher ratio associated with increased risk of heart disease and stroke Fatty Acyl-CoA Desaturase Introduces Double Bonds into Fatty Acids to Form Desaturated Fatty Acids - Palmitate (16:0) and stearate (18:0) serve as precursors of the most common monounsaturated fatty acids, palmitoleate (16:1) and oleate (18:1) - Double bonds are introduced into the acyl chain by an oxidative reaction catalysed by fatty acyl-CoA desaturase (don’t need to know this reaction) Acetyl-CoA is Converted to Malonyl-CoA by Acetyl-CoA Carboxylase Recall glucagon stimulates triacylglycerol catabolism and fatty acid release in fat cells (see last lecture) Acetyl-CoA Carboxylase (ACC) is a Key Regulator of Fatty Acid Metabolism Blocking Fatty Acid Synthesis Reduces Lung Cancer Growth Fatty Acid and Lipid Synthesis: Summary Triacylglycerol and Phospholipids: Revision Types of membrane lipids: - Phospholipids (glycerophospholipids, sphingolipids) - Glycolipids (sphingolipids, galactolipids) - Sterols (cholesterol; next lecture) Triacylglycerol and Phospholipid Synthesis: Revision Triacylglycerols and Phospholipids Share Common Precursors - Glycerol-3-phosphate and two fatty acyl-CoAs - Glycerol-3-phosphate results from glyceroneogenesis - Fatty acyl-CoAs are formed from free fatty acids by acyl-CoA synthetases Seen fatty acyl-CoA and acyl-CoA synthetase before? Fatty acid degradation (transport of fatty acids into mitochondria) - These precursors combine to form phosphatidic acid (phosphatidate) - Phosphatidic acid is present in trace amounts but is an essential intermediate in lipid synthesis Triacylglycerol and Phospholipid Synthesis Fatty Acid Synthesis (don’t need to know, just use as summary) Glycerophospholipids: Revision - Most abundant lipid in membranes - Comprised of glycerol with two fatty acid chains and a head-group alcohol - Named after the head-group alcohol (don’t need to know details) Triacylglycerol and Phospholipid Synthesis Glycerophospholipid Synthesis (don’t need to know, just use as summary) Sphingolipids: Revision - Highest expression in cells of the central nervous system - Comprised of a sphingosine backbone, long-chain fatty acid and a polar head group - Three sub-classes of sphingolipids: - Glycosphingolipids - Gangliosides - Sphingomyleins (don’t need to know details) Sphingolipid Synthesis (don’t need to know, just use as summary) only neeed to knwo what’s in red) Triacylglycerol and Phospholipid Synthesis: Summary Appendix Case Review - Laura Ambrose is a 6-year-old female who was brought to the emergency department by her parents Laura is experiencing the following: - 4- to 5-day history of nausea, vomiting and abdominal pain - Drowsy - Breathing deeply and quickly - Thirsty and urinating frequently - Weight loss Laura is diagnosed with type 1 diabetes - Physical Examination: Ketotic Breath - Finger Prick Test: Ketones - high - Urine Dipstick Test: Ketones ++ Glucose ++ What are ketones? All three molecules soluble in blood and urine Where are Ketones Produced and Found? Ketones Bodies are a Useful Source of Energy for the Brain Why Ketones Cannot be Used in the Liver Acetoacetate and D-β-hydroxybutyrate → 2x acetyl CoA → Citric acid cycle - No enzyme for catabolism in liver Where do ketones come from? 4 Key Metabolic Pathways: - Gluconeogenesis - β-oxidation - Citric Acid Cycle - Electron Transport Chain Gluconeogenesis - Glucose: exported as fuel for brain and tissue Beta Oxidation For fatty acid breakdown Electron Transport Chain Where Do Ketones Come From? How do Ketones Relate to Diabetes? How Does This Relate to Diabetes? - Low insulin and ↓ glucose uptake → Citric acid cycle intermediates used for gluconeogenesis → Citric acid cycle impaired - Glucose not taken up by liver → ↑ malonyl-CoA → ↑ activation of carnitine acyltransferase I → ↑ β-oxidation of fatty acids → ↑ NADH and acetyl-CoA → Impaired citric acid cycle - Excess NADH and ↓ citric acid cycle intermediates → Impaired citric acid cycle → Excess acetyl-CoA forms ketone bodies → Ketotic breath, ketonemia, ketonuria What is Ketosis? In individuals with uncontrolled diabetes, acetone, acetoacetate and D-β-hydroxybutyrate are produced in excess in the liver What is Happening in Uncontrolled Diabetes? - Increased acetoacetate and D-b-hydroxybutyrate - Overwhelmed bicarbonate buffering capacity → lower blood pH - Ketoacidosis - Lethargy, coma, death Summary - Ketone bodies in healthy individuals are used as fuel in extra-hepatic tissues, including the brain. - Ketone bodies are formed from acetyl-CoA following the β-oxidation of fatty acids. - In uncontrolled diabetes, beta-oxidation increases and the citric acid cycle decreases, resulting in excess acetyl-CoA and ketone body formation. - The excess ketone body formation results in ketotic breath, and high amounts of ketones in the blood and urine as seen in Laura and others with uncontrolled diabetes. Ketone Body, Lipid Hormone and Vitamin Synthesis (all structures in this lecture are optional information unless stated otherwise) Lipid and Fatty Acid Degradation and Ketone Body Synthesis: Revision Triacylglycerol and Phospholipid Synthesis: Revision Eicosanoid Synthesis Eicosanoid Hormones - Arachidonate (arachidonic acid) derivatives - Act locally and are short lived Four classes: - Prostaglandins: muscle contraction, hormone responsiveness, increase body temperature, induce pain and inflammation - Thromboxanes: blood clot formation, reduce blood flow - Leukotrienes: muscle contraction, inflammation, allergic reactions - Lipoxins: resolution of inflammation Palmitate Serves as a Precursor of Long-chain Fatty Acids: Revision - Palmitate is lengthened by the fatty acid elongation system on the endoplasmic reticulum membrane - Coenzyme A (rather than ACP) is the acyl carrier - Mammals lack the ability to introduce double bonds beyond carbon position 9 - Thus, mammals require essential omega fatty acids in their diet Phospholipases Target Specific Sites of Phospholipids (know the general structure and phospholipase sites) Eicosanoids are Derived from Arachidonic Acid via Phospholipase A2 Prostaglandins and Thromboxanes are Synthesised by Cyclooxygenase, which is Impaired by Non-steroidal AntiInflammatory Drugs (NSAIDs) (be able to label) Leukotrienes and Lipoxins are Synthesised by Lipooxygenase (be able to label) Synthesis Cholesterol and its Derivatives Cholesterol is the Main Sterol in Animals Main sterols: - Cholesterol (animals) - Stigmasterol (plants) - Ergosterol (fungi) - Absent in bacteria Sterols contain a steroid nucleus - Almost planar - Relatively rigid - Polar Sterols serve as membrane constituents, and precursors of steroid hormones and bile salts. Stigmasterol and other plant derivates can reduce cholesterol and can be used as food additives to help achieve this. Cholesterol is Essential for Life - Bad reputation due to its association with cardiovascular disease - Essential molecule in animals (but not required in diet) - Synthesised from the simple precursor acetate (from acetyl-CoA) Cholesterol Synthesis HMG-CoA Reductase is the Essential Control Point in Cholesterol Synthesis Pathway (should know these enzymes) - Seen HMG-CoA synthase before? Ketone body synthesis - HMG-CoA reductase is the target of cholesterol lowering compounds called statins (eg: Atarvastatin or Lipitor) - Statins are the most widely prescribed lipid-lowering medication in Australia (and worldwide) Squalene is a Common Precursor of Sterols in Different Kingdoms - Liver comprises 25-30% of shark mass - Squalene helps sharks maintain their buoyancy - Squalene may provide health benefits (anti-oxidant in skin, anti-cancer agent and pro-immune agent) Cholesterol Has Various Fates Bile Acids Emulsify Dietary Lipids: Revision - Bile acids are made from cholesterol and conjugated to glycine or taurine to form bile salts in the liver - In the small intestine, bile salts emulsify dietary lipids into micelles (lipid droplets) making the triacylglycerols more accessible to digestive enzymes to aid their digestion Cholesterol and Other Lipids Are Carried on Plasma Lipoproteins: Revision Steroid Hormones - Lack alkyl chain attached to fourth (D) ring - More polar than cholesterol - Travel through bloodstream on protein carriers - Bind to highly specific receptors to induces changes in gene expression and metabolism Steroid Hormones are Derived from Cholesterol Cholesterol Has Various Fates Vitamins Water soluble vitamins (Bs, C) - Vitamin Bs function as coenzymes and/or give rise to energy carriers - Vitamin C functions as a reducing agent and as an antioxidant Lipid soluble vitamins are involved in many processes but do not function as coenzymes - Vitamin A functions as a pigment and in gene regulation - Vitamin D involved in calcium and phosphate metabolism - Vitamin E is an important antioxidant - Vitamin K is a blood-clotting factor Different Forms of Energy Carriers: Revision - ATP (adenosine triphosphate) - Energy release following transfer of groups - NADH (nicotinamide adenine dinucleotide) - NADPH (nicotinamide adenine dinucleotide phosphate) - FADH2 (flavin adenine dinucleotide) - All three release high energy electrons and H+ - Coenzyme A (CoA) - Carrier of acyl (R−C=O) and acetyl (CH3−C=O) groups - Energy release from high energy bond of the acyl (acetyl) group - Others (guanosine triphosphate, carboxylated biotin, uridine diphosphate glucose, Sadenosylmethionine) Some Coenzymes are Derived from Group B Vitamins (don’t need to know details) Vitamin D Synthesis - 7-Dehydrocholersterol, formed from cholesterol, is converted to biologically inactive vitamin D3 in the skin by sunlight - Vitamin D3 sequentially is converted to biologically active 1a,25-dihydroxyvitamin D3 in the liver then kidney 1a,25-Dihydroxyvitamin D3 and Bone Metabolism - Regulates calcium and phosphate metabolism to promote bone metabolism - Deficiency impairs bone mineralisation leading to bone disease: - Rickets: bowed legs in children - Osteomalacia: pelvic and leg pain and increased risk of bone fractures in adults - Deficiency linked to neurodegenerative, cardiovascular and autoimmune diseases, and infection - Due to poor dietary intake and/or reduced sun exposure (eg: skin cancer prevention programs, certain ethnic groups) Lipid Hormone Synthesis: Summary