BiochemistrySlides2020.pdf

DNA Structure Jason Ryan, MD, MPH DNA Contains genetic code Nucleus of eukaryotic cells Cytoplasm of prokaryotic cells Wikipedia/Public Domain DNA Structure Sugar (ribose) backbone Nitrogenous base Phosphate bonds Wikiped...

DNA Structure Jason Ryan, MD, MPH DNA Contains genetic code Nucleus of eukaryotic cells Cytoplasm of prokaryotic cells Wikipedia/Public Domain DNA Structure Sugar (ribose) backbone Nitrogenous base Phosphate bonds Wikipedia/Public Domain DNA Vocabulary Nucleotide/Nucleoside Nitrogenous base Purine/Pyrimidine Nucleotides DNA: Polymer Nucleotide: Monomer Pentose sugar Nitrogenous base Phosphate group Ribonucleotide Deoxyribonucleotide Binhtruong/Wikipedia  AfraTafreeh.com for more Nucleoside vs. Nucleotide Adenosine Nucleotide Monophosphate Nitrogenous base Sugar Phosphate group Nucleoside Base and sugar No phosphate group Wikipedia/Public Domain  Nitrogenous Bases Pyrimidines Cytosine Thymine Uracil Purines Adenine Guanine Nucleotides Cytidine Thymidine Uridine Adenosine Guanosine Nucleotides Synthesized as monophosphates Converted to triphosphate form Added to DNA Deoxyadenosine Triphosphate  AfraTafreeh.com for more Base Pairing DNA Adenine-Thymine Guanine-Cytosine RNA Adenine-Uracil Guanine-Cytosine More C-G bonds = ↑ Melting temperature Wikipedia/Public Domain DNA Methylation Methyl group added to cytosine Occurs in segments with CG patterns (“CG islands”) Both strands Inactivates transcription (“epigenetics”) Human DNA: ~70% methylated Unmethylated CG stimulate immune response Cytosine 5-methylcytosine Bacterial DNA Methylation Bacteria methylate cytosine and adenine Methylation protects bacteria from viruses (phages) Non-methylated DNA destroyed by endonucleases “Restriction-modification systems” Wikipedia/Public Domain Chromatin Found in nucleus of eukaryotic cells DNA plus proteins = chromatin Chromatin condenses into chromosomes Nucleosome Key protein: Histones Units of histones plus DNA = nucleosomes H2A, H2B, H3, H4 Wikipedia/Public Domain  AfraTafreeh.com for more H2A, H2B, H3, H4 Histones Peptides H1, H2A, H2B, H3, H4 Contain basic amino acids High content of lysine, arginine Wikipedia/Public Domain Positively charged Binds negatively charged phosphate backbone H1 distinct from others Not in nucleosome core Larger, more basic Ties beads on string together DNA Structure DNA DNA Beads on H1 plus a string Condensation Histones Richard Wheeler/Wikipedia Drug-Induced Lupus Fever, joint pains, rash after starting drug Anti-histone antibodies (>95% cases) Contrast with anti-dsDNA in classic lupus Classic drugs: Hydralazine Procainamide Isoniazid Chromatin Types Heterochromatin Condensed Gene sequences not transcribed (varies by cell) Significant DNA methylation Euchromatin Less condensed Transcription Significant histone acetylation  Acetyl Histone Acetylation Group Lysine Acetylation Acetyl group added to lysine Relaxes chromatin for transcription Deacetylation Reverse effect Annabelle L. Rodd, Katherine Ververis, and Tom C. Karagiannis Epigenetics Histone Acetylation Transcription Transcription DNA Methylation Histone deacetylase inhibitors HDACs Potential therapeutic effects Anti-cancer Increased expression of HDACs some tumors Huntington’s disease Movement disorder Abnormal huntingtin protein Gain of function mutation (mutant protein) Possible mechanism: histone deacetylation → gene silencing Leads to neuronal cell death in striatum Dokmanovic et al. Histone deacetylase inhibitors: overview and perspectives Mol Cancer Res. 2007 Oct;5(10):981-9. Purine Metabolism Jason Ryan, MD, MPH  Nucleotides Pyrimidines Cytidine Thymidine Uridine Purines Adenosine Guanosine Nucleotide Roles RNA and DNA monomers Energy: ATP Physiologic mediators cAMP levels → blood flow cGMP → second messenger  AfraTafreeh.com for more Sources of Nucleotides Diet (exogenous) Biochemical synthesis (endogenous) Direct synthesis Salvage Key Points Ribonucleic acids (RNA) synthesized first RNA converted to deoxyribonucleic acids (DNA) Different pathways for purines versus pyrimidines All nitrogen comes from amino acids Purine Synthesis Goal is to create AMP and GMP Ingredients: Ribose phosphate (HMP Shunt) Amino acids Carbons (tetrahydrofolate, CO2) Adenosine Guanosine Purine Synthesis Step 1: Create PRPP 5-Phosphoribosyl-1-pyrophosphate Ribose 5-phosphate (PRPP) Purine Synthesis Hypoxanthine Step 2: Create IMP Amino Acids Folate CO2 5-Phosphoribosyl-1-pyrophosphate Inosine monophosphate (PRPP) (IMP) Purine Synthesis N Two rings with two nitrogens: N 6 unit, 3 double bonds 6 5 N 5 unit, 2 double bonds N Adenine Guanine Hypoxanthine Purine Synthesis N N Nitrogen Sources 6 5 N N Glycine Aspartate N N N N Glutamine Purine Synthesis N N Carbon Sources 6 5 N CO2 N Glycine N Tetrahydrofolate N N N Tetrahydrofolate *Key Point Folate contributes to formation of purines Purine Synthesis Step 3: Create AMP and GMP Adenosine-MP Inosine monophosphate (IMP) Guanosine-MP Purine Synthesis Summary Starts with ribose phosphate from HMP shunt Key intermediates are PRPP and IMP AMP 5-Ribose PRPP IMP Phosphate GMP Aspartate Glycine Glutamine THF CO2  AfraTafreeh.com for more Purine Synthesis Regulation IMP/AMP/GMP - Glutamine-PRPP amidotransferase AMP 5-Ribose PRPP IMP Phosphate GMP Deoxyribonucleotides ADP Ribonucleotide Reductase dADP GDP dGDP Purine Synthesis Drugs & Diseases Ribavirin (antiviral) Inhibits IMP dehydrogenase Blocks conversion IMP to GMP Inhibits synthesis guanine nucleotides (purines) Mycophenolate (immunosuppressant) Inhibits IMP dehydrogenase Purine Fates Adenine Guanine Hypoxanthine Uric Acid Salvage Excretion Purine Salvage Salvages bases: adenine, guanine, hypoxanthine Converts back into nucleotides: AMP, GMP, IMP Requires PRPP 5-Phosphoribosyl-1-pyrophosphate (PRPP) Purine Salvage Hypoxanthine and Guanine Hypoxanthine PRPP Inosine monophosphate HGPRT (IMP) Hypoxanthine-Guanine phosphoribosyltransferase Guanine Guanosine-MP PRPP Purine Salvage Adenine Adenine Adenosine-MP APRT Adenine PRPP phosphoribosyltransferase Purine Salvage Drugs & Diseases 6-Mercaptopurine Hypoxanthine Guanine Chemotherapy agent Mimics hypoxanthine/guanine Added to PRPP by HGPRT → Thioinosinic acid Inhibits multiple steps in de novo synthesis ↓IMP/AMP/GMP + PRPP 6-MP Purine Salvage Drugs & Diseases Azathioprine Immunosuppressant Converted to 6-MP Azathioprine 6-MP Purine Breakdown Xanthine Hypoxanthine Oxidase Xanthine Oxidase Xanthine Uric Acid Guanase Guanine  Purine Breakdown *SCID Adenosine Deaminase Adenosine-MP Adenosine Inosine APRT Purine nucleoside phosphorylase Adenine Hypoxanthine Purine Salvage Xanthine Oxidase Drugs & Diseases Gout Hypoxanthine Uric Acid Excess uric acid Crystal deposition in joints → pain, swelling, redness Can occur from overproduction of uric acid High cell turnover (trauma, chemotherapy) Consumption of purine-rich foods (meat, seafood) Treatment: inhibit xanthine oxidase (allopurinol) James Heilman, MD/Wikipedia Purine Salvage Drugs & Diseases Azathioprine and 6-MP Metabolized by xanthine oxidase Caution with allopurinol May boost effects May increase toxicity Xanthine Oxidase Thiouric acid 6-MP (inactive) Purine Salvage Drugs & Diseases Lesch-Nyhan syndrome X-linked absence of HGPRT Excess uric acid production (“juvenile gout”) Excess de novo purine synthesis (↑PRPP, ↑IMP) Neurologic impairment (mechanism unclear) Hypotonia, chorea Classic feature: self mutilating behavior (biting, scratching) Can treat hyperuricemia Limited treatments for neurologic features Classic presentation Male child with motor symptoms, self-mutilation, gout Purine Salvage Drugs & Diseases syndrome X-linked absence of HGPRT Excess uric acid production (“juvenile gout”) Excess de novo purine synthesis (↑PRPP, ↑IMP) Neurologic impairment (mechanism unclear) Hypotonia, chorea Classic feature: self mutilating behavior (biting, scratching) No treatment Classic presentation Male child with motor symptoms, self-mutilation, gout Purine Metabolism Summary Torres RJ, Puig JG/Wikipedia Pyrimidine Metabolism Jason Ryan, MD, MPH  Nucleotides Pyrimidines Cytidine Thymidine Uridine Purines Adenosine Guanosine Pyrimidine Synthesis Goal is to create CMP, UMP, TMP Ingredients: Ribose phosphate (HMP Shunt) Amino acids Carbons (tetrahydrofolate, CO2) Cytidine Thymidine Uridine Pyrimidine Synthesis Step 1: Make carbamoyl phosphate Note: ring formed first then ribose sugar added ATP ADP Glutamine CO2 Carbamoyl Phosphate Carbamoyl phosphate synthetase II UTP Pyrimidine Synthesis Step 2: Make orotic acid Aspartate Carbamoyl Phosphate Orotic Acid Pyrimidine Synthesis Step 3: Make UMP UMP Synthase Orotic Acid Uridine-MP 5-Phosphoribosyl-1-pyrophosphate (PRPP) Key Point UMP synthesized first CMP, TMP derived from UMP CMP Carbamoyl Orotic Glutamine UMP Phosphate Acid TMP UMP Synthase Bifunctional Pyrimidine Ring Two nitrogens/four carbons C Cytosine Carbamoyl Phosphate N C Aspartate Uracil C C N Thymine Pyrimidine Synthesis Drugs and Diseases Orotic aciduria Autosomal recessive Defect in UMP synthase Buildup of orotic acid Loss of pyrimidines Orotic Acid Pyrimidine Synthesis Drugs and Diseases Key findings Orotic acid in urine Megaloblastic anemia Orotic Acid No B12/folate response Growth retardation Treatment: Uridine Bypasses UMP synthase Megaloblastic Anemia Wikipedia/Public Domain Ornithine transcarbamoylase OTC Key urea cycle enzyme Combines carbamoyl phosphate with ornithine Makes citrulline OTC deficiency → increased carbamoyl phosphate ↑ carbamoyl phosphate → ↑ orotic acid Don’t confuse with orotic aciduria Both have orotic aciduria OTC only: ↑ ammonia levels (urea cycle dysfunction) Ammonia → encephalopathy (baby with lethargy, coma) Cytidine Uridine-MP Uridine-TP Cytidine-TP ATP Pyrimidine Synthesis Drugs and Diseases Ara-C (Cytarabine or cytosine arabinoside) Chemotherapy agent Converted to araCTP Mimics dCTP (pyrimidine analog) Inhibits DNA polymerase H Ara-C dCytidine Thymidine Only used in DNA Deoxythymidine is only required nucleotide Synthesized from deoxyuridine Thymidine Uridine Thymidine Step 1: Convert UMP to dUDP Uridine-DP deoxyuridine-DP Uridine-MP Ribonucleotide Reductase Pyrimidine Synthesis Drugs and Diseases Hydroxyurea Inhibits ribonucleotide reductase Blocks formation of deoxynucleotides (RNA intact!) Rarely used for malignancy Can be used for polycythemia vera, essential thrombocytosis Used in sickle cell anemia Causes increased fetal hemoglobin levels (mechanism unclear) Thymidine Step 2: Convert dUDP to dUMP 1 Carbon added Step 3: Convert dUMP to dTMP Thymidylate Synthase deoxyuridine-MP (dUMP) deoxythymidine-MP (dTMP) Thymidine Thymidylate Synthase dUMP dTMP Source of 1 carbon N5, N10 Tetrahydrofolate Folate Compounds Folate Dihydrofolate Tetrahydrofolate Folate Compounds Tetrahydrofolate N5, N10 Tetrahydrofolate Thymidine Thymidylate Synthase dUridine-MP Thymidine-MP DHF Folate N5, N10 Tetrahydrofolate Dihydrofolate THF Reductase * Folate = 1 carbon carriers Pyrimidine Synthesis Drugs and Diseases 5-FU Chemotherapy agent Mimics uracil Converted to 5-FdUMP (abnormal dUMP) Covalently binds N5,N10 TFH and thymidylate synthase Result: inhibition thymidylate synthase Blocks dTMP synthesis (“thymineless death”) Uracil Pyrimidine Synthesis Drugs and Diseases Methotrexate Chemotherapy agent, immunosuppressant Mimics DHF Inhibits dihydrofolate reductase Blocks synthesis dTMP Rescue with leucovorin (folinic acid; converted to THF) Folate Methotrexate Pyrimidine Synthesis Drugs and Diseases Sulfonamides antibiotics Bacteria cannot absorb folic acid Synthesize THF from para-aminobenzoic acid (PABA) Sulfonamides mimic PABA Block THF synthesis ↓ THF formation → ↓ dTMP (loss of DNA synthesis) No effect human cells (dietary folate) Fdardel/Wikipedia Bacterial THF Synthesis PABA Dihydropteroate Sulfonamides Synthase Dihydropteroic Acid Dihydrofolic Acid Dihydrofolate Trimethoprim Reductase THF DNA Pyrimidine Synthesis Drugs and Diseases Folate deficiency Main effect: loss of dTMP production → ↓ DNA production RNA production relatively intact (does not require thymidine) Macrocytic anemia (fewer but larger RBCs) Neural tube defects in pregnancy  Vitamin B12 Thymidylate Synthase dUridine-MP Thymidine-MP DHF Folate N5, N10 Tetrahydrofolate N5 Methyl THF Dihydrofolate THF Reductase B12 Vitamin B12 Required to regenerate THF from N5-Methyl THF Deficiency = “Methyl folate trap” Loss of dTMP synthesis (megaloblastic anemia) Neurological dysfunction (demyelination)  Homocysteine and MMA Folate N5-Methyl THF THF B12 Homocysteine Methionine Methylmalonic Acid (MMA) Methymalonyl CoA B12 Succinyl CoA B12 versus Folate Deficiency Homocysteine Both folate and B12 required to covert to methionine Elevated homocysteine in both deficiencies Methylmalonic Acid B12 also converts MMA to succinyl CoA B12 deficiency = ↑ methylmalonic acid (MMA) level Folate deficiency = normal MMA level B12 versus Folate Deficiency Folate B12 RBC ↓ ↓ MCV ↑ ↑ Homocysteine ↑ ↑ Methylmalonic acid (MMA) -- ↑ Megaloblastic Anemia Anemia (↓Hct) Large RBCs (↑MCV) Hypersegmented neutrophils Commonly caused by defective DNA production Folate deficiency B12 (neuro symptoms, MMA) Orotic aciduria Drugs (MTX, 5-FU, hydroxyurea) Zidovudine (HIV NRTIs) Wikipedia/Public Domain Glucose Jason Ryan, MD, MPH Carbs Carbohydrate = “watered carbon” Most have formula Cn(H2O)m Glucose C6H12O6 Wikipedia/Public Domain  AfraTafreeh.com for more Carbs Monosaccharides (C6H12O6) Glucose, Fructose, Galactose Glucose Carbs Disaccharides = 2 monosaccharides Broken down to monosaccharides in GI tract Lactose (galactose + glucose); lactase Sucrose (fructose + glucose); sucrase Lactose Complex Carbs Polysaccharides: polymers of monosaccharides Starch Plant polysaccharide (glucose polymers) Glycogen Animal polysaccharide (also glucose polymers) Cellulose Plant polysaccharide of glucose molecules Different bonds from starch Cannot be broken down by animals “Fiber” in diet → improved bowel function Glucose All carbohydrates broken down into: Glucose Fructose Galactose  Glucose Metabolism Glucose Anaerobic TCA Cycle HMP Shunt Fatty Acid Glycogenesis Metabolism Synthesis Ribose/ H2O/CO2 Glycogen Lactate NADPH Fatty Acids Glucose Metabolism Liver Most varied use of glucose TCA cycle for ATP Glycogen synthesis Glucose Metabolism Brain Constant use of glucose for TCA cycle (ATP) Little glycogen storage Muscle/heart TCA cycle (ATP) Transport into cells heavily influenced by insulin More insulin → more glucose uptake Store glucose as glycogen Glucose Metabolism Red blood cells No mitochondria Use glucose for anaerobic metabolism (make ATP) Generate lactate Also use glucose for HMP shunt (NADPH) Adipose tissue Mostly converts glucose to fatty acids Like muscle, uptake influenced by insulin Glucose Entry into Cells Na+ independent entry 14 different transporters described GLUT-1 to GLUT-14 Varies by tissue (i.e. GLUT-1 in RBCs) Na+ dependent entry Glucose absorbed from low → high concentration Intestinal epithelium Renal tubules ↑[Glucose] GLUT ↓[Glucose] Glucose GI Absorption GI Lumen Interstitium/Blood 2 Na+ SGLT 1 Glucose Na+ GLUT 2 Glucose ATP Na+ Proximal Tubule Lumen (Urine) Interstitium/Blood Na+ Na+ ATP K+ Glucose Glucose Glucose Entry into Cells GLUT-1 Insulin independent (uptake when [glucose] high) Brain, RBCs GLUT-4 Insulin dependent Fat tissue, skeletal muscle GLUT-2 Insulin independent Bidirectional (gluconeogenesis) Liver, kidney Intestine (glucose OUT of epithelial cells to portal vein) Pancreas Glycolysis Jason Ryan, MD, MPH Glycolysis Used by all cells of the body Sequence of reactions that occurs in cytoplasm Converts glucose (6 carbons) to pyruvate (3 carbons) Generates ATP and NADH NADH Nicotinamide adenine dinucleotide Two nucleotides Carries electrons NAD+ Accepts electrons NADH Donates electrons Can donate to electron transport chain → ATP  AfraTafreeh.com for more Glycolysis Glucose Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-bisphosphate Glyceraldehyde-3-phosphate Dihydroxyacetone Phosphate 1,3-bisphosphoclycerate 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate Pyruvate Glycolysis Priming Stage Uses energy (consumes 2 ATP) First and last reactions most critical Glucose ATP Glucose-6-phosphate Fructose-6-phosphate ATP Fructose-1,6-bisphosphate Hexokinase vs. Glucokinase Hexokinase Glucose Found in most tissues ATP Strongly inhibited by G6P - Blocks cells from hording glucose ADP Insulin = no effect Low Km (usually operates max) Glucose-6-phosphate Low Vm (max is not that high) Hexokinase V = Vm* [S] Km + [S] Vmax Hexokinase Low Km Quickly Reach Vm V Vm low [S] Hexokinase vs. Glucokinase Glucokinase Glucose Found in liver and pancreas ATP NOT inhibited by G6P - Induced by insulin ADP Insulin promotes transcription Inhibited by F6P (overcome by ↑glucose) Glucose-6-phosphate High Km (rate varies with glucose) Fructose-6-phosphate *Enzyme inactive when (1) low glucose and (2) high F6P Glucokinase High Vm liver after meals V = Vm* [S] Vmax Km + [S] Activity varies with [glucose] Glucokinase High Km High Vm V Sigmoidal Curve Cooperativity [S] Glucokinase regulatory protein (GKRP) Translocates glucokinase to nucleus Result: inactivation of enzyme Fructose 6 phosphate: GKRP binds glucokinase → nucleus (inactive) Glucose: Competes with GKRP for GK binding Glucokinase → cytosol (active) GK F-6-P GKRP Nucleus GKRP GK Glucose Hexokinase vs. Glucokinase Low blood sugar Hexokinase working (no inhibition G6P) Glucokinase inactive (rate α glucose; low insulin) Glucose to tissues, not liver Glucose High blood sugar ATP Hexokinase inactive (inhibited by G6P) Glucokinase working (high glucose, high insulin) ADP Liver will store glucose as glycogen Glucose-6-phosphate Fructose-6-phosphate Glucokinase Deficiency Results in hyperglycemia Pancreas less sensitive to glucose Mild hyperglycemia Often exacerbated by pregnancy Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762. Phosphofructokinase-1 Rate limiting step for glycolysis Consumes 2nd ATP in priming stage Irreversible Commits glucose to glycolysis HMP shunt, glycogen synthesis no long possible Fructose-6-phosphate ATP ADP Fructose-1,6-bisphosphate Regulation of Glycolysis Phosphofructokinase-1 Key inhibitors (less glycolysis) Citrate (TCA cycle) ATP Key inducers (more glycolysis) AMP Fructose 2,6 bisphosphate (insulin) Fructose-6-phosphate ATP ADP Fructose-1,6-bisphosphate Fructose 2,6 Bisphosphate Regulation of Glycolysis PFK2 F-2,6-bisphosphate Fructose-6-phosphate Fructose 1,6 Bisphosphatase2 PFK1 Fructose 1,6 Bisphosphatase1 Fructose-1,6-bisphosphate On/off switch glycolysis ↑ = glycolysis (on) ↓ = no glycolysis (gluconeogenesis) Insulin and Glucagon I G PFK2/FBPase2 P PFK2/FBPase2 Fructose 2,6 Bisphosphate Regulation of Glycolysis Fed State Insulin ↑Insulin PFK2 ↑F 2,6 BP ↑ F 2,6 BP F6P F2,6BPase + F 1,6 BP Pixabay Fructose 2,6 Bisphosphate Regulation of Glycolysis Fasting State Glucagon ↓Insulin ↓F 2,6 BP PFK2 ↓ F 2,6 BP F6P F2,6BPase P - F 1,6 BP Aude/Wikipedia Glycolysis Splitting Stage Fructose 1,6-phosphate to two molecules GAP Reversible for gluconeogenesis Fructose-1,6-bisphosphate Glyceraldehyde-3-phosphate Dihydroxyacetone Phosphate Glycolysis Energy Stage Glyceraldehyde-3-phosphate NAD+ Starts with GAP NADH 1,3-bisphosphoclycerate Two ATP per GAP ATP 3-phosphoglycerate Total per glucose = 4 2-phosphoglycerate Anaerobic Metabolism (no O2) Phosphoenolpyruvate 2 ATP (net) ATP Pyruvate NADH NAD+ Lactate TCA Cycle Glycolysis Energy Stage Phosphoenolpyruvate Pyruvate kinase Not reversible Pyruvate Inhibited by ATP, alanine ATP Kinase Activated by fructose 1,6 BP Pyruvate “Feed forward” activation Glucagon/epinephrine Phosphorylation Inactivation of pyruvate kinase Slows glycolysis/favors gluconeogenesis Alanine Cycle Skeletal muscles can degrade protein for energy Produce alanine → blood → liver Liver converts alanine to glucose Glucose/ Glucose Glycogen Urea Pyruvate Pyruvate Amino Acids Alanine Alanine Alanine transaminase (ALT) Liver Muscle Glycolysis Energy Stage Phosphoenolpyruvate Lactate dehydrogenase (LDH) Pyruvate Kinase Pyruvate → Lactate ATP Plasma elevations common Pyruvate Hemolysis LDH Myocardial infarction Some tumors Pleural effusions Lactate NAD+ Acetyl-CoA Transudate vs. exudate TCA Cycle NADH Glyceraldehyde-3-phosphate NAD+ Limited supply NAD+ NADH 1,3-bisphosphoclycerate Must regenerate 3-phosphoglycerate O2 present NADH → NAD (mitochondria) 2-phosphoglycerate O2 absent NADH → NAD+ via LDH Phosphoenolpyruvate Pyruvate NADH NAD+ Lactate TCA Cycle Lactic Acidosis ↓O2 → ↓ pyruvate entry into TCA cycle ↑ lactic acid production ↓pH, ↓HCO3- Elevated anion gap acidosis Sepsis, bowel ischemia, seizures Lactate Pyruvate Dehydrogenase Lactate TCA Cycle Muscle Cramps Too much exercise → too much NAD consumption Exceed capacity of TCA cycle/electron transport Elevated NADH/NAD ratio Favors pyruvate → lactate pH falls in muscles → cramps Distance runners: lots of mitochondria (bigger, too) Pyruvate Kinase Deficiency Autosomal recessive disorder RBCs most effected No mitochondria Databese Center for Life Science (DBCLS) Require PK for anaerobic metabolism Loss of ATP Membrane failure → phagocytosis in spleen Usually presents as newborn Extravascular hemolysis Splenomegaly Disease severity ranges based on enzyme activity 2,3 Bisphosphoglycerate Created from diverted 1,3 BPG Glyceraldehyde-3-phosphate Used by RBCs 2,3 BPG 1,3-bisphosphoglycerate No mitochondria No TCA cycle BPG ATP Mutase 3-phosphoglycerate Sacrifices ATP from glycolysis 2-phosphoglycerate 2,3 BPG alters Hgb binding Phosphoenolpyruvate ATP Pyruvate Lactate TCA Cycle Databese Center for Life Science (DBCLS) Right Curve Shifts Easier to release O2 100 Hb % Saturation 75 Right Shift 50 ↑BPG (Also ↑ Co2, Temp, H+) 25 25 50 75 100 pO2 (mmHg) Energy Yield from Glucose ATP generated depends on cells/oxygen Highest yield with O2 and mitochondria Allows pyruvate to enter TCA cycle Converts pyruvate/NADH → ATP Blausen.com staff. "Blausen gallery 2014". Wikiversity Journal of Medicine. DOI:10.15347/wjm/2014.010. ISSN 20018762 Energy from Glucose Oxygen and Mitochondria Glucose + 6O2 → 32/30 ATP + 6CO2 + 6 H2O 32 ATP = malate-aspartate shuttle (liver, heart) 30 ATP = glycerol-3-phosphate shuttle (muscle) No Oxygen or No Mitochondria Glucose → 2 ATP + 2 Lactate + 2 H2O *RBCs = no mitochondria Summary Glucose Key Steps Glucose-6-phosphate Regulation Fructose-6-phosphate #1: Hexokinase/Glucokinase AMP F2,6BP #2: PFK1 Fructose-1,6-bisphosphate #3: Pyruvate Kinase Irreversible Glyceraldehyde-3-phosphate Glucose → G6P (Hexo/Glucokinase) 1,3-bisphosphoclycerate F6P → F 1,6 BP (PFK1) PEP → pyruvate (pyruvate kinase) 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate ATP Alanine Pyruvate Summary ATP Glucose Key Steps Glucose-6-phosphate ATP expended Fructose-6-phosphate Glucose → G6P ATP F6P → F1,6BP Fructose-1,6-bisphosphate ATP generated 1,3BPG → 3PG Glyceraldehyde-3-phosphate PEP → pyruvate 1,3-bisphosphoclycerate ATP 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate ATP Pyruvate Gluconeogenesis Jason Ryan, MD, MPH Gluconeogenesis Glucose Glucose-6-phosphate Glucose from other carbons Fructose-6-phosphate Sources of glucose Pyruvate Fructose-1,6-bisphosphate Lactate Amino acids Glyceraldehyde-3-phosphate Propionate (odd chain fats) 1,3-bisphosphoglycerate Glycerol (fats) 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate Pyruvate  Liver Glucose Glucose Liver Pyruvate Alanine Cori Cycle Cycle Alanine Lactate Gluconeogenesis Acetyl-Coa TCA Cycle Pyruvate Pyruvate Pyruvate Carboxylase ↓ATP ↑ATP Gluconeogenesis Acetyl-Coa *Pyruvate carboxylase inactive without Acetyl-Coa ATP TCA Cycle Gluconeogenesis Step #1: Pyruvate → Phosphoenolpyruvate ATP CO2 GTP Pyruvate Oxaloacetate Phosphoenolpyruvate Pyruvate (OAA) PEP (PEP) Carboxylase Carboxykinase Biotin Gluconeogenesis Step #1: Pyruvate → Phosphoenolpyruvate Mitochondria Cytosol Malate Shuttle Pyruvate Oxaloacetate Phosphoenolpyruvate Pyruvate (OAA) PEP (PEP) Carboxylase Carboxykinase Biotin Cofactor for carboxylation enzymes All add 1-carbon group via CO2 Pyruvate carboxylase Acetyl-CoA carboxylase Propionyl-CoA carboxylase Deficiency Very rare (vitamin widely distributed) Massive consumption raw egg whites (avidin) Dermatitis, glossitis, loss of appetite, nausea Pyruvate Carboxylase Deficiency Very rare Presents in infancy with failure to thrive Elevated pyruvate → lactate Lactic acidosis Gluconeogenesis Step #2: Fructose 1,6 bisphosphate → Fructose 6 phosphate Rate limiting step Fructose-6-phosphate Phosphofructokinase-1 Fructose 1,6 bisphosphatase 1 Fructose-1,6-bisphosphate Gluconeogenesis Fructose-6-phosphate Phosphofructokinase-1 Fructose 1,6 bisphosphatase 1 Fructose-1,6-bisphosphate ATP AMP Fructose 2,6 bisphosphate  Fructose 2,6 Bisphosphate Regulation of Glycolysis/Gluconeogenesis PFK2 Fructose-2,6-bisphosphate Fructose-6-phosphate Fructose 1,6 Bisphosphatase 2 PFK1 Fructose 1,6 Bisphosphatase 1 Fructose-1,6-bisphosphate On/off switch glycolysis ↑ = glycolysis (on) ↓ = no glycolysis (gluconeogenesis) Fructose 2,6 Bisphosphate Regulation of Gluconeogenesis Levels rise with high insulin (fed state) Levels fall with high glucagon (fasting state) Drives glycolysis versus gluconeogenesis PFK1 vs. F 1,6 BPtase1 Phosphofructokinase-1 Fructose 1,6 Bisphosphatase Glycolysis Gluconeogenesis AMP AMP F 2,6, Bisphosphate F 2,6, Bisphosphate ATP ATP Citrate Fructose-6-phosphate PFK1 Fructose 1,6 Bisphosphate1 Fructose-1,6-bisphosphate Gluconeogenesis Step #3: Glucose 6-phosphate → Glucose Occurs mainly in liver and kidneys Other organs shunt G6P → glycogen Glucose-6 Glucose Phosphate Glucose-6 Phosphatase Endoplasmic Reticulum  Gluconeogenesis Glucose Glucose-6-phosphate F2,6BP Insulin/ Fructose-6-phosphate Glucagon AMP ATP Fructose-1,6-bisphosphate Phosphoenolpyruvate Acetyl CoA Pyruvate Hormonal Control Glucagon + Glucose Glucose-6-phosphate Glucagon Fructose-6-phosphate (↓F2,6BP) + Fructose-1,6-bisphosphate Phosphoenolpyruvate Glucagon Pyruvate x Gluconeogenesis Substrates Glucose Fructose-1,6-bisphosphate Glyceraldehyde-3-phosphate Dihydroxyacetone Phosphate Odd Chain Fatty Acids PEP Proprionyl OAA CoA Amino Glycerol Lactate Pyruvate Acids Alanine Hormones Insulin Shuts down gluconeogenesis (favors glycolysis) Action via F 2,6, BP Glucagon (opposite of insulin) Other Hormones Epinephrine Raises blood glucose Gluconeogenesis and glycogen breakdown Cortisol Increases gluconeogenesis enzymes Hyperglycemia common side effect steroid drugs Thyroid hormone Increases gluconeogenesis Glycogen Jason Ryan, MD, MPH Glycogen Storage form of glucose Polysaccharide Repeating units of glucose Lin Mei/Flikr Most abundant in muscle, liver Muscle: glycogen for own use Liver: glycogen for body Wikipedia/Public Domain Glycogen Wikipedia/Public Domain Boumphreyfr/Wikipedia Glycogen Synthesis Glucose ATP Hexokinase/ Glucokinase ADP Glycogen Glucose-6-phosphate Glycolysis Glycogen Synthesis Glucose-6-phosphate Glucose-1-phosphate UDP-glucose UTP pyrophosphorylase UDP-Glucose Glycogen Synthase Unbranched Glycogen Branching Enzyme Branched Glycogen  Lin Mei/Flikr Glycogen Breakdown Glucose-6 Phosphatase Glucose Glucose-6-phosphate Glycolysis Glucose-1-phosphate α1,4 glucosidase (lysosomes) Glycogen UDP-Glucose phosphorylase Unbranched Glycogen (α1,4) Debranching Enzyme Branched Glycogen (α1,6) Glycogen Breakdown Phosphorylase Removes glucose molecules from glycogen polymer Creates glucose-1-phosphate Stops when glycogen branches decreased to 2-4 linked glucose molecules (limit dextrins) Stabilized by vitamin B6 Debranching enzyme Cleaves limit dextrins Debranching Enzyme Hormonal Regulation Glycogen Glucagon Insulin Epinephrine Glucose Hormonal Regulation Glycogen Glucagon Insulin Epinephrine Enzyme Phosphorylation P P Glycogen Glycogen Phosphorylase Synthase Glucose Hormonal Regulation Glycogen Glucagon Insulin Epinephrine Glycogen Glycogen Phosphorylase Synthase Glucose Epinephrine and Glucagon Glycogen Phosphorylase Epi Gluc Adenyl + Cyclase + P Glycogen Glycogen cAMP Phosphorylase Breakdown P Glycogen Phosphokinase A PKA Insulin Glycogen Phosphorylase I Tyrosine Kinase Glycogen Glycogen Phosphorylase Breakdown Protein Phosphatase 1 P GPKinase A P Protein Phosphatase 1 GPKinase A Epinephrine and Glucagon Glycogen Synthase Epi Gluc Adenyl + Cyclase + cAMP Inhibition Glycogen Synthase P Glycogen Synthesis Glycogen PKA Synthase Insulin Glycogen Synthase I Tyrosine Kinase Protein Phosphatase 1 P Glycogen Synthase P Protein Phosphatase 1 Glycogen Glycogen Synthesis Synthase Muscle Contraction Glycogen Phosphorylase P Glycogen Glycogen Phosphorylase Breakdown Calcium/Calmodulin GPKinase A Glycogen Regulation Glycogen ATP Glucose Glucose 6-P Glycogen Glycogen Phosphorylase Synthase AMP Glucose Glycogen as Fuel Ingested Glucose Glucose g/hr Wikipedia/Public Domain Glycogen Gluconeogenesis (proteins/fatty acids) 0 8 16 24 36 Hours Glycogen Storage Diseases Most autosomal recessive Defective breakdown of glycogen Liver: hypoglycemia Muscle: weakness More than 14 described Von Gierke’s Disease Glycogen Storage Disease Type I Glucose-6-phosphatase deficiency (Type Ia) Type Ib: Glucose transporter deficiency Presents in infancy: 2-6 months of age Severe hypoglycemia between meals Lethargy Seizures Lactic acidosis (Cori cycle) Enlarged liver (excess glycogen) Can lead to liver failure Cori Cycle Lactate Cycle Petaholmes/Wikipedia Von Gierke’s Disease Glycogen Storage Disease Type I Diagnosis: DNA testing (preferred) Liver biopsy (historical test) Treatment: Cornstarch (glucose polymer) Avoid sucrose, lactose, fructose, galactose Feed into glycolysis pathways Cannot be metabolized to glucose via gluconeogenesis Worsen accumulation of glucose 6-phosphate Pompe’s Disease Glycogen Storage Disease Type II Acid alpha-glucosidase deficiency Also “lysosomal acid maltase” Accumulation of glycogen in lysosomes Classic form presents in infancy Severe disease → often death in infancy/childhood Pompe’s Disease Glycogen Storage Disease Type II Enlarged muscles Cardiomegaly Enlarged tongue Hypotonia Liver enlargement (often from heart failure) No metabolic problems (hypoglycemia) Death from heart failure Cori’s Disease Glycogen Storage Disease Type III Debranching enzyme deficiency Similar to type I except: Milder hypoglycemia No lactic acidosis (Cori cycle intact) Muscle involvement (glycogen accumulation) Key point: Gluconeogenesis is intact Cori’s Disease Glycogen Storage Disease Type III Classic presentation: Infant or child with hypoglycemia/hepatomegaly Hypotonia/weakness Possible cardiomyopathy with hypertrophy McArdle’s Disease Glycogen Storage Disease Type V Muscle glycogen phosphorylase deficiency Myophosphorylase deficiency Skeletal muscle has unique isoform of G-phosphorylase Glycogen not properly broken down in muscle cells Usually presents in adolescence/early adulthood Exercise intolerance, fatigue, cramps Poor endurance, muscle swelling, and weakness Myoglobinuria and CK release (especially with exercise) Urine may turn dark after exercise Glycogen Synthase Deficiency Can’t form liver glycogen normally Fasting hypoglycemia with ketosis Postprandial hyperglycemia May present in older children (less frequent feeds) Morning fatigue Symptoms improve with food HMP Shunt Jason Ryan, MD, MPH HMP Shunt Series of reactions that goes by several names: Hexose monophosphate shunt Pentose phosphate pathway 6-phosphogluconate pathway Glucose 6-phosphate “shunted” away from glycolysis  Glucose HMP Shunt Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-bisphosphate Glyceraldehyde-3-phosphate 1,3-bisphosphoclycerate 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate Pyruvate HMP Shunt Synthesizes: NADPH (many uses) Ribose 5-phosphate (nucleotide synthesis) Two key clinical correlations: G6PD deficiency Thiamine deficiency (transketolase) HMP Shunt All reactions occur in cytosol Two phases: Oxidative: irreversible, rate-limiting Reductive: reversible NADPH Glucose Ribulose-5 Phosphate Glucose-6-phosphate Ribose-5 Fructose-6-phosphate Phosphate HMP Shunt Oxidative Reactions Glucose-6 Phosphate Dehydrogenase Glucose CO2 Ribulose-5 6 phospho- Phosphate gluconolactone Glucose-6-phosphate NADPH NADP+ NADPH NADP+ HMP Shunt Reductive Reactions NADPH Glucose Ribulose-5 Phosphate Glucose-6-phosphate Ribose-5 Fructose-6-phosphate Phosphate Transketolase Transketolase Transfers a carbon unit to create F-6-phosphate Requires thiamine (B1) as a co-factor Wernicke-Korsakoff syndrome Abnormal transketolase may predispose Affected individuals may have abnormal binding to thiamine Ribose-5-Phosphate Purine Nucleotides Adenosine, Guanosine Pyrimidine Nucleotides Ribose 5-phosphate Cytosine, Uridine, Thymidine NADPH Nicotinamide adenine dinucleotide phosphate Similar structure to NADH Not used for oxidative phosphorylation (ATP) NADH NADPH NADPH Uses Used in “reductive” reactions Releases hydrogen to form NADP+ Use #1: Co-factor in fatty acid, steroid synthesis Liver, mammary glands, testis, adrenal cortex Use #2: Phagocytosis Use #3: Protection from oxidative damage Respiratory Burst Phagocytes generate H2O2 to kill bacteria “Oxygen dependent” killing “Oxygen independent”: low pH, enzymes Uses three key enzymes: NADPH oxidase Superoxide dismutase Myeloperoxidase Respiratory Burst O2 NADPH NADPH Oxidase NADP+ O2- Superoxide Dismutase Bacterial H2O2 Death Cl- Myeloperoxidase HOCl CGD Chronic Granulomatous Disease Loss of function of NADPH oxidase Phagocytes cannot generate H2O2 Catalase (-) bacteria generate their own H2O2 Phagocytes use despite enzyme deficiency Catalase (+) bacteria breakdown H2O2 Host cells have no H2O2 to use → recurrent infections Five organisms cause almost all CGD infections: Staph aureus, Pseudomonas, Serratia, Nocardia, Aspergillus Source: UpToDate G6PD Deficiency Glucose-6-Phosphate Dehydrogenase NADPH required for normal red blood cell function H2O2 generation triggered in RBCs Infections Drugs Fava beans Need NADPH to degrade H2O2 Absence of required NADPH → hemolysis Glutathione Erythrocytes Trigger Databese Center for Life Science (DBCLS) H2O2 Glutathione NADP+ Glutathione Glutathione Peroxidase Reductase H2O Glutathione NADPH + H+ HMP Shunt Disulfide Requires G6PD G6PD Deficiency Glucose-6-Phosphate Dehydrogenase X-linked disorder (males) Most common human enzyme disorder High prevalence in Africa, Asia, the Mediterranean May protect against malaria Recurrent hemolysis after exposure to trigger May present as dark urine Other HMP functions usually okay Nucleic acids, fatty acids, etc. G6PD Deficiency Glucose-6-Phosphate Dehydrogenase Classic findings: Heinz bodies and bite cells Heinz bodies: oxidized Hgb precipitated in RBCs Bite cells: phagocytic removal by splenic macrophages Heinz bodies Bite cells G6PD Deficiency Triggers Infection: Macrophages generate free radicals Fava beans: Contain oxidants Drugs: Antibiotics (sulfa drugs, dapsone, nitrofurantoin, INH) Anti-malarials (primaquine, quinidine) Aspirin, acetaminophen (rare) G6PD Deficiency Diagnosis and Treatment Diagnosis: Fluorescent spot test Detects generation of NADPH from NADP Positive test if blood spot fails to fluoresce under UV light Treatment: Avoidance of triggers Fructose and Galactose Jason Ryan, MD, MPH Fructose and Galactose Isomers of glucose (same formula: C6H12O6) Galactose (and glucose) taken up by SGLT1 Na+ dependent transporter Fructose taken up by facilitated diffusion GLUT-5 All leave enterocytes by GLUT-2 Carbohydrate GI Absorption GI Lumen Interstitium/Blood 2 Na+ SGLT 1 Glucose Glucose Galactose GLUT Galactose 2 Fructose GLUT 5 Fructose Na+ ATP Na+  AfraTafreeh.com for more Fructose Commonly found in sucrose (glucose + fructose) Glucose Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-bisphosphate Glyceraldehyde- Dihydroxyacetone 3-phosphate Phosphate Glyceraldehyde Fructose-1-Phosphate Fructose  Fructose Glyceraldehyde- Dihydroxyacetone 3-phosphate Phosphate Triokinase Aldolase B ATP Glyceraldehyde Fructose-1-Phosphate Fructose Fructokinase (liver)  Fructose Glucose Special Point Glucose-6-phosphate Phosphofructokinase-1 Rate-limiting step: glycolysis Fructose-6-phosphate Fructose-1,6-bisphosphate Glyceraldehyde-3-phosphate Dihydroxyacetone Phosphate Fructose bypasses PFK-1 Rapid metabolism 1,3-bisphosphoclycerate 3-phosphoglycerate Fructose-1-Phosphate 2-phosphoglycerate Phosphoenolpyruvate Fructose Pyruvate  Fructose Glucose Special Point Glucose-6-phosphate Hexokinase Initial enzyme glycolysis Fructose Fructose-6-phosphate Hexokinase can metabolize Fructose-1,6-bisphosphate small amount of fructose Glyceraldehyde-3-phosphate Dihydroxyacetone Phosphate 1,3-bisphosphoclycerate 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate Pyruvate Essential Fructosuria Deficiency of fructokinase Benign condition Fructose not taken up by liver cells Fructose appears in urine (depending on intake) Hereditary Fructose Intolerance Deficiency of aldolase B Build-up of fructose 1-phosphate Depletion of ATP Hereditary Fructose Intolerance ↑Fructose-1-Phosphate ↓ATP ↓Gluconeogenesis ↓Glycogen Breakdown Hypoglycemia/ Hepatomegaly Liver Failure Vomiting Hereditary Fructose Intolerance Baby just weaned from breast milk Failure to thrive Symptoms after feeding Hypoglycemia (seizures) Enlarged liver Part of newborn screening panel Treatment: Avoid fructose, sucrose, sorbitol Polyol Pathway Glucose → Fructose NADPH NADP+ NAD+ NADH Glucose Sorbitol Fructose Aldose Sorbitol Reductase Dehydrogenase Galactose Commonly found in lactose (glucose + galactose) Converted to glucose 6-phosphate Galactose Galactose 1-Phosphate Glucose 1-Phosphate Glycogen Glucose Glucose 6-Phosphate Glycolysis Galactose Galactose ATP Galactokinase Galactose 1-Phosphate UDP-Glucose Galactose 1-Phosphate Uridyltransferase (GALT) Glucose 1-Phosphate Classic Galactosemia Deficiency of galactose 1-phosphate uridyltransferase Autosomal recessive disorder Galactose-1-phosphate accumulates in cells Leads to accumulation of galactitol in cells Polyol Pathway NADPH NADP+ NAD+ NADH Glucose Sorbitol Fructose Sorbitol Dehydrogenase Aldose Reductase Galactose Galactitol Classic Galactosemia Presents in infancy Often first few days of life Shortly after consumption of milk Wikipedia/Public Domain Liver accumulation galactose/galactitol Liver failure Jaundice Hepatomegaly Failure to thrive Cataracts if untreated Wikipedia/Public Domain Classic Galactosemia Screening: GALT enzyme activity assay Treatment: avoid galactose Galactokinase Deficiency Milder form of galactosemia Galactose not taken up by cells Accumulates in blood and urine Main problem: cataracts as child/young adult May present as vision problems Wikipedia/Public Domain Pyruvate Dehydrogenase Jason Ryan, MD, MPH Pyruvate End product of glycolysis Liver Glucose Glucose Liver Pyruvate Alanine Cori Cycle Cycle Alanine Lactate Gluconeogenesis Acetyl-Coa Pyruvate Transported into mitochondria for: Entry into TCA cycle Gluconeogenesis Outer membrane: a voltage-gated porin complex Inner: mitochondrial pyruvate carrier (MPC) Blausen gallery 2014". Wikiversity Journal of Medicine Pyruvate Pyruvate Pyruvate Pyruvate Carboxylase Dehydrogenase Complex Gluconeogenesis Acetyl-Coa ATP TCA Cycle Pyruvate Dehydrogenase Complex Complex of 3 enzymes Pyruvate dehydrogenase (E1) Dihydrolipoyl transacetylase (E2) Dihydrolipoyl dehydrogenase (E3) Requires 5 co-factors NAD+ FAD Coenzyme A (CoA) E1 Thiamine Lipoic acid E3 E2 Pyruvate Dehydrogenase Complex O CO2 CH3-C-COO- Pyruvate E1 OH Thiamine-PP CH3-CH-TPP CoA NADH NAD E2 Acetyl-CoA E3 Lipoic Acid O CH3-C-Lipoic Acid FAD Thiamine PDH Cofactors Vitamin B1 Converted to thiamine pyrophosphate (TPP) Co-factor for four enzymes Pyruvate dehydrogenase α-ketoglutarate dehydrogenase (TCA cycle) α-ketoacid dehydrogenase (branched chain amino acids) Transketolase (HMP shunt) Thiamine ATP AMP Thiamine pyrophospate Thiamine Deficiency ↓ production of ATP ↑ aerobic tissues affected most (nerves/heart) Beriberi Underdeveloped areas Dry type: polyneuritis, muscle weakness Wet type: tachycardia, high-output heart failure, edema Wernicke-Korsakoff syndrome Alcoholics (malnourished, poor absorption vitamins) Confusion, confabulation Thiamine and Glucose Malnourished patients: ↓glucose ↓thiamine If glucose given first → unable to metabolize Case reports of worsening Wernicke-Korsakoff FAD PDH Cofactors Synthesized from riboflavin (B2) Added to adenosine → FAD Accepts 2 electrons → FADH2 Riboflavin Flavin Adenine Dinucleotide NAD+ PDH Cofactors Carries electrons as NADH Niacin Synthesized from niacin (B3) Niacin: synthesized from tryptophan Used in electron transport Nicotinamide Adenine Dinucleotide Coenzyme A PDH Cofactors Also a nucleotide coenzyme (NAD, FAD) Synthesized from pantothenic acid (B5) Accepts/donates acyl groups Pantothenic Acid Coenzyme A Acetyl-CoA B Vitamins * All water soluble * All wash out quickly from body B1: Thiamine (not stored in liver like B12) B2: Riboflavin (FAD) B3: Niacin (NAD) B5: Pantothenic Acid (CoA) Ragesoss/Wikipedia Lipoic Acid PDH Cofactors Bonds with lysine → lipoamide Co-factor for E2 Wikipedia/Public Domain Inhibited by arsenic Poison (metal) Binds to lipoic acid → inhibits PDH (like thiamine deficiency) Oxidized to arsenous oxide: smells like garlic (breath) Non-specific symptoms: vomiting, diarrhea, coma, death PDH Regulation PDH Kinase: phosphorylates enzyme → inactivation PDH phosphatase: dephosphorylation → activation ↑NAD/NADH P ↑ADP Ca2+ PDH PDH ↓NAD/NADH ↑ACoA ↑ATP Active Inactive PDH Complex Deficiency Rare inborn error of metabolism Pyruvate shunted to alanine, lactate Often X linked Most common cause: mutations in PDHA1 gene Codes for E1-alpha subunit E1 α PDH Complex Deficiency Key findings (infancy): Poor feeding Growth failure Developmental delays Labs: Elevated alanine Lactic acidosis Wikipedia/Public Domain Mitochondrial Disorders Inborn error of metabolism All cause severe lactic acidosis Key examples: Pyruvate dehydrogenase complex deficiency Pyruvate carboxylase deficiency Cytochrome oxidase deficiencies PDH Complex Deficiency Treatment Thiamine, lipoic acid (optimize remaining PDH) Ketogenic diet Low carbohydrates (reduces lactic acidosis) High fat Ketogenic amino acids: Lysine and leucine Drives ketone production (instead of glucose) Ketogenic Amino Acids Acetoacetate Leucine Acetyl-CoA Lysine TCA Cycle Jason Ryan, MD, MPH TCA Cycle Tricarboxylic Acid Cycle, Krebs Cycle, Citric Acid Cycle Metabolic pathway Converts acetyl-CoA → CO2 Derives energy from reactions TCA Cycle Tricarboxylic Acid Cycle, Krebs Cycle, Citric Acid Cycle All reactions occur in mitochondria Produces: NADH, FADH2 → electron transport chain (ATP) GTP CO2 Blausen gallery 2014". Wikiversity Journal of Medicine  TCA Cycle Acetyl-CoA NADH Oxaloacetate Citrate Malate Isocitrate CO2 NADH Fumarate α-ketoglutarate FADH2 Succinate Succinyl-CoA CO2 NADH GTP Citrate Synthesis 6 Carbon structure Oxaloacetate (4C) + Acetyl-CoA (2C) Inhibited by ATP Special Points: Inhibits PFK1 (glycolysis) Activates ACoA carboxylase Acetyl-CoA (fatty acid synthesis) Citrate Oxaloacetate Synthase Citrate CoA ATP Fasting State Oxaloacetate used for gluconeogenesis ↓ oxaloacetate for TCA cycle Acetyl-CoA (fatty acids) → Ketone bodies Pyruvate Acetyl-CoA Ketones Glucose Citrate Oxaloacetate Synthase Citrate CoA Isocitrate Isomer of citrate Enzyme: aconitase Forms intermediate (cis-aconitate) then isocitrate Inhibited by fluoroacetate: rat poison Citrate Isocitrate α-Ketoglutarate Rate limiting step of TCA cycle Inhibited by: ATP NADH Activated by: Isocitrate ADP Isocitrate CO2 Ca++ Dehydrogenase NADH α-ketoglutarate Succinyl-CoA α-ketoglutarate dehydrogenase complex Similar to pyruvate dehydrogenase complex Cofactors: Thiamine CoA Succinyl-CoA NAD NADH FADH α-ketoglutarate Lipoic acid CoA α-KG Dehydrogenase Ca++ Succinyl-CoA CO2 NADH Succinate Succinyl-CoA synthetase Succinate Succinyl-CoA CoA GTP Fumarate Succinate dehydrogenase Unique enzyme: embedded mitochondrial membrane Functions as complex II electron transport Succinate FAD Complex II Electron Fumarate Succinate FADH2 Transport Dehydrogenase Fumarate Also produced several other pathways Urea cycle Purine synthesis (formation of IMP) Amino acid breakdown: phenylalanine, tyrosine Malate and Oxaloacetate NADH Oxaloacetate Malate dehydrogenase Malate Fumarase Fumarate Malate Shuttle Malate “shuttles” molecules cytosol → mitochondria Key points: Malate can cross mitochondrial membrane (transporter) NADH and oxaloacetate cannot cross Two key uses: Transfer of NADH into mitochondria Transfer of oxaloacetate OUT of mitochondria NADH Oxaloacetate Malate dehydrogenase Malate Malate Shuttle Use #1: Transfer of NADH Aspratate OAA Malate Glut NADH NAD+ Cytosol α-KG Mitochondria Aspratate OAA Malate α-KG NADH NAD+ Glut Malate Shuttle Use #2: Transfer of oxaloacetate Gluconeogenesis OAA Malate NADH NAD+ Cytosol Mitochondria OAA Malate NADH NAD+ TCA Intermediates Fatty Amino Acids Glucose Acids Oxaloacetate Citrate Malate Isocitrate Amino Acids Fumarate α-ketoglutarate Succinate Succinyl-CoA Succinyl CoA Odd Chain Fatty Acids Branched Chain Amino Acids TCA Cycle (α-KG) Methylmalonyl CoA Succinyl-CoA TCA Cycle Heme (succinate) Synthesis TCA Cycle Key Points Inhibited by: ATP NADH Acetyl CoA Citrate Succinyl CoA  TCA Cycle Pyruvate Dehydrogenase Acetyl-CoA Pyruvate ATP, Acetyl-CoA, NADH NADH Citrate synthase Oxaloacetate Citrate ATP ATP NADH Malate Citrate Isocitrate Isocitrate Dehydrogenase CO2 NADH NADH Fumarate Succinyl CoA α-ketoglutarate α-KG FADH2 Dehydrogenase Succinate Succinyl-CoA CO2 NADH GTP TCA Cycle Key Points Activated by: ADP Calcium  TCA Cycle Acetyl-CoA Pyruvate NADH Oxaloacetate Citrate ADP Malate Ca++ Isocitrate Isocitrate Dehydrogenase CO2 NADH Fumarate Ca++ α-ketoglutarate α-KG FADH2 Dehydrogenase Succinate Succinyl-CoA CO2 NADH GTP Electron Transport Chain Jason Ryan, MD, MPH Electron Transport Chain NADH FADH2 Blausen gallery 2014". Wikiversity Journal of Medicine  Aerobic Metabolism Cytosol Mitochondria GTP NADH NADH 2 ATP Acetyl CoA ATP NADH Pyruvate FADH2 Glucose Pyruvate NADH NADH 2 NADH Acetyl CoA ATP NADH FADH2 GTP Malate Shuttle Aspartate OAA Malate α-KG Glut NADH NAD+ Cytosol Mitochondria Aspartate OAA Malate α-KG NADH NAD+ Glut Glycerol Phosphate Shuttle Glycerol Phosphate Dehydrogenase Dihydroxyacetone Glycerol phosphate Phosphate NADH NAD+ Cytosol Glycerol Glycerol Phosphate Phosphate Dehydrogenas Dehydrogenase e Mitochondria FAD FADH2 Electron Transport Extract electrons from NADH/FADH2 Transfer to oxygen (aerobic respiration) In process, generate/capture energy NADH → NAD+ + H+ + 2e- FADH2 → FAD + 2 H+ + 2e- 2e- + 2H+ + ½O2 → H2O Blausen gallery 2014". Wikiversity Journal of Medicine  Electron Transport Complexes Cytosol Outer Membrane Inter Membrane Space Inner I II III IV Membrane Complex I NADH Dehydrogenase Oxidizes NADH (NADH → NAD+) Transfers electrons to coenzyme Q (ubiquinone) e- NADH CoQ Complex I CoQ shuttles electrons to complex III Pumps H+ into intermembrane space Key intermediates: Flavin mononucleotide (FMN) Iron sulfur compounds (FeS) e- e- e- NADH FMN FeS CoQ  Electron Transport Cytosol Outer Membrane Inter Membrane H+ e- e- Space CoQ Inner I III Membrane H+ CoQ 10 Supplements Some data indicate statins decrease CoQ levels Hypothesized to contribute to statin myopathy CoQ 10 supplements may help in theory No good data to support this use Ragesoss/Wikipedia Complex II Succinate dehydrogenase (TCA cycle) Electrons from succinate → FADH2 → CoQ FAD Succinate II FADH2 CoQ Fumarate Succinate Dehydrogenase Complex III Cytochrome bc1 complex Transfers electrons CoQ → cytochrome c Pumps H+ to intermembrane space  Electron Transport Cytosol Outer Membrane Inter Membrane H+ e- Cyt c e- Space Inner III IV Membrane H+ Cytochromes Class of proteins Contains a heme group Iron plus porphyrin ring Hgb: mostly Fe2+ Cytochromes: Fe2+ → Fe3+ Oxidation state changes with electron transport Electron transport: a, b, c Cytochrome P450: drug metabolism Complex IV Cytochrome a + a3 Cytochrome c oxidase (reacts with oxygen) Contains copper (Cu) Electrons and O2 → H2O Also pumps H+  Electron Transport Cytosol Outer Membrane Inter H+ H+ H+ H+ Membrane H+ H+ H +H+ H+ H H + H+ + H+ H+ H+ H+H+ H+ + H H+ + Space HH+ + H + H+ + H+ H H + H + H+H H + H + H + H + H + H + H+ H+ H+ H+ H + CoQ Cyt c Inner I II III IV Membrane O2 H2O Phosphorylation Two ways to produce ATP: Substrate level phosphorylation Oxidative phosphorylation Substrate level phosphorylation (via enzyme): Phosphoenolpyruvate ADP ATP Pyruvate  Oxidative Phosphorylation Cytosol Outer Membrane Inter H+ H+ H+ H+ H+ H+ H+ Membrane H+ H+ H+ H+ H + + + H+ H+ H+ H+ H+ H H+ H Space Inner ATP Membrane Synthase ADP ATP ATP Synthase Complex V Converts proton (charge) gradient → ATP “electrochemical gradient” “proton motive force” Protons move down gradient (“chemiosmosis”) P/O Ratio ATP per molecule O2 Classically had to be an integer 3 per NADH 2 per FADH2 Newer estimates 2.5 per NADH 1.5 per FADH2 Hinkle P. P/O ratios of mitochondrial oxidative phosphorylation. Biochimica et Biophysica Act 1706 (Jan 2005) 1-11  Aerobic Energy Production 30/32 ATP Cytosol Mitochondria per glucose GTP (1ATP) NADH NADH 2 ATP Acetyl CoA 9 ATP NADH Pyruvate NADH (2.5) FADH2 Glucose Pyruvate NADH (2.5) NADH NADH 2 NADH Acetyl CoA 9 ATP NADH Malate 2 NADH (5) FADH2 2 FADH2 (3) GTP (1 ATP) Glycerol-3-P Drugs and Poisons Two ways to disrupt oxidative phosphorylation #1: Block/inhibit electron transport #2: Allow H+ to leak out of inner membrane space “Uncoupling” of electron transport/oxidative phosphorylation Inhibitors Rotenone (insecticide) Binds complex I Prevents electron transfer (reduction) to CoQ Antimycin A (antibiotic) Complex III (bc1 complex) Complex IV Carbon monoxide (binds a3 in Fe2+ state – competes with O2) Cyanide (binds a3 in Fe3+ state) Cyanide Poisoning CNS: Headache, confusion Cardiovascular: Initial tachycardia, hypertension Respiratory: Initial tachypnea Bright red venous blood: ↑O2 content Almond smell Anaerobic metabolism: lactic acidosis Mullookkaaran/Wikipedia Cyanide Poisoning Nitroprusside: treatment of hypertensive emergencies Contains five cyanide groups per molecule Toxic levels with prolonged infusions Treatment: Nitrites (amyl nitrite) Converts Fe2+ → Fe3+ in Hgb (methemoglobin) Fe3+ in Hgb binds cyanide, protects mitochondria  Uncoupling Agents Cytosol Outer Membrane Inter H+ H+ H+ H+ H+ H+ H+ Membrane H+ H+ H+ H+ H + + + H+ H+ H+ H+ H+ H H+ H Space Inner ATP Membrane Synthase ADP ATP Uncoupling Agents 2,4 dinitrophenol (DNP) Aspirin (overdose) Brown fat Newborns (also hibernating animals) Uncoupling protein 1 (UCP-1, thermogenin) Sympathetic stimulation (NE, β receptors) → lipolysis Electron transport → heat (not ATP) All lead to production of heat Pixabay/Public Domain Oligomycin A Macrolide antibiotic Inhibits ATP synthase Protons cannot move through enzyme Protons trapped in intermembrane space Oxidative phosphorylation stops ATP cannot be generated Fatty Acids Jason Ryan, MD, MPH Lipids Mostly carbon and hydrogen Not soluble in water Many types: Fatty acids Triacylglycerol (triglycerides) Cholesterol Phospholipids Steroids Glycolipids Lipids Glycerol Fatty Acid Triglyceride Fatty Acid and Triglycerides Most lipids degraded to free fatty acids in intestine Enterocytes convert FAs to triacylglycerol Chylomicrons carry through plasma TAG degraded back to free fatty acids Lipoprotein lipase Endothelial surfaces of capillaries Abundant in adipocytes and muscle tissue Vocabulary “Saturated” fat (or fatty acid) Contains no double bonds “Saturated” with hydrogen Usually solid at room temperature Raise LDL cholesterol “Unsaturated” fat Contains at least one double bond “Monounsaturated:” One double bond “Polyunsaturated:” More than one double bond More Vocabulary Trans fat Double bonds (unsaturated) can be trans or cis Most natural fats have cis configuration Trans from partial hydrogenation (food processing method) Can increase LDL, lower HDL Omega-3 fatty acids Type of polyunsaturated fat Found in fish oil Lower triglyceride levels eicosapentaenoic acid (EPA) Fatty Acid Metabolism Fatty acids synthesis Liver, mammary glands, adipose tissue (small amount) Excess carbohydrates and proteins → fatty acids Fatty acid storage Adipose tissue Stored as triglycerides Fatty acid breakdown β-oxidation Acetyl CoA → TCA cycle → ATP Fatty Acid Synthesis In high energy states (fed state): Lots of acetyl-CoA Lots of ATP Inhibition of isocitrate dehydrogenase (TCA cycle) Result: High citrate level Acetyl-CoA ATP Isocitrate Citrate Dehydrogenase Oxaloacetate Synthase Citrate CoA Fatty Acid Synthesis Step 1: Citrate to cytosol via citrate shuttle Key point: Acetyl-CoA cannot cross membrane Citrate Cytosol Mitochondria Acetyl-CoA ATP Isocitrate Citrate Dehydrogenase Oxaloacetate Synthase Citrate CoA Fatty Acid Synthesis Step 2: Citrate converted to acetyl-CoA Net effect: Excess acetyl-CoA moved to cytosol ATP-Citrate Lyase Citrate Acetyl-CoA CoA ATP ADP Oxaloacetate Fatty Acid Synthesis Step 3: Acetyl-CoA converted to malonyl-CoA Rate limiting step Glucagon Insulin Epinephrine β-oxidation - Citrate + - Acetyl-CoA Carboxylase Acetyl-CoA Malonyl-CoA CO2 Biotin Daniel W. Foster. Malonyl CoA: the regulator of fatty acid synthesis and oxidation J Clin Invest. 2012;122(6):1958–1959. Biotin Cofactor for carboxylation enzymes All add 1-carbon group via CO2 Pyruvate carboxylase Acetyl-CoA carboxylase Propionyl-CoA carboxylase Fatty Acid Synthesis Step #4: Synthesis of palmitate Enzyme: fatty acid synthase Uses carbons from acetyl CoA and malonyl CoA Creates 16 carbon fatty acid Requires NADPH (HMP Shunt) Palmitate Fatty Acid Storage Palmitate can be modified to other fatty acids Used by various tissues based on needs Stored as triacylglycerols in adipose tissue Fatty Acid Breakdown Key enzyme: Hormone sensitive lipase Removes fatty acids from TAG in adipocytes Activated by glucagon and epinephrine Fatty Acid Breakdown Fatty Acid Hormone Sensitive Lipase Triacylglycerol Liver Glycerol Glycerol Glucose Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-bisphosphate Glyceraldehyde-3-phosphate Dihydroxyacetone Phosphate 1,3-bisphosphoglycerate Glycerol-3-Phosphate Dehydrogenase 3-phosphoglycerate Glycerol-3-Phosphate 2-phosphoglycerate Glycerol Kinase Phosphoenolpyruvate Pyruvate Glycerol Fatty Acid Breakdown Fatty acids transported via albumin Taken up by tissues Not used by: RBCs: Glycolysis only (no mitochondria) Brain: Glucose and ketones only Databese Center for Life Science (DBCLS) Wikipedia/Public Domain Fatty Acid Breakdown β-oxidation Removal of 2-carbon units from fatty acids Produces acetyl-CoA, NADH, FADH2 β-oxidation Step #1: Convert fatty acid to fatty acyl CoA ATP CoA Fatty Fatty acyl Acid CoA R—C—OH Long Chain R—C—CoA O Fatty Acyl CoA O synthetase β-oxidation Step #2: Transport fatty acyl CoA → inner mitochondria Uses carnitine shuttle Carnitine in diet Also synthesized from lysine and methionine Only liver, kidney can synthesize de novo Muscle and heart depend on diet or other tissues Carnitine  Carnitine Shuttle Malonyl-CoA Fatty acyl Cytosol CoA - Carnitine Palmitoyl Inner Transferase-1 Membrane Space Fatty acyl Acyl CoA Carnitine Carnitine R—C—CoA R—C—Carnitine O O Mitochondrial Matrix R—C—CoA O CoA Fatty acyl CPT-2 Acyl CoA Carnitine Carnitine Deficiencies Several potential secondary causes Malnutrition Liver disease Increased requirements (trauma, burns, pregnancy) Hemodialysis (↓ synthesis; loss through membranes) Major consequence: Inability to transport LCFA to mitochondria Accumulation of LCFA in cells Low serum carnitine and acylcarnitine levels Carnitine Deficiencies Muscle weakness, especially during exercise Cardiomyopathy Hypoketotic hypoglycemia when fasting Tissues overuse glucose Poor ketone synthesis without fatty acid breakdown Primary systemic carnitine deficiency Mutation affecting carnitine uptake into cells Infantile phenotype presents first two year of life Encephalopathy Hepatomegaly Hyperammonemia (liver dysfunction) Hypoketotic hypoglycemia Low serum carnitine: kidneys cannot resorb carnitine Reduced carnitine levels in muscle, liver, and heart β-oxidation Step #3: Begin “cycles” of beta oxidation Removes two carbons Shortens chain by two Generates NADH, FADH2, Acetyl CoA β carbon CoA-S α carbon β-oxidation First step in a cycle involves acyl-CoA dehydrogenase Adds a double bond between α and β carbons β carbon CoA-S FAD α carbon Acyl-CoA dehydrogenase FADH2 CoA-S Acyl-CoA Dehydrogenase Family of four enzymes Short Medium Long Very-long chain fatty acids Well described deficiency of medium chain enzyme MCAD Deficiency Medium Chain Acyl-CoA Dehydrogenase Autosomal recessive disorder Poor oxidation 6-10 carbon fatty acids Severe hypoglycemia without ketones Dicarboxylic acids 6-10 carbons in urine High acylcarnitine levels Dicarboxylic Acid MCAD Deficiency Medium Chain Acyl-CoA Dehydrogenase Gluconeogenesis shutdown Pyruvate carboxylase depends on Acetyl-CoA Acetyl-CoA levels low in absence β-oxidation Exacerbated in fasting/infection Treatment: Avoid fasting  Odd Chain Fatty Acids β-oxidation proceeds until 3 carbons remain Proprionyl-CoA → Succinyl-CoA → TCA cycle Key point: Odd chain FA → glucose Succinyl-CoA S-CoA Propionyl-CoA Carboxylase (Biotin) Propionyl CoA  Propionyl CoA Common pathway to TCA cycle Elevated methylmalonic acid seen in B12 deficiency Biotin Propionyl CoA MethylMalonyl-CoA Succinyl-CoA B12 Amino Acids Odd Chain Isoleucine Fatty Acids TCA Cycle Valine Threonine Cholesterol Methionine Methylmalonic Acidemia Deficiency of Methylmalonyl-CoA mutase Anion gap metabolic acidosis CNS dysfunction Often fatal early in life Methylmalonyl-CoA mutase Methylmalonyl-CoA Succinyl-CoA B12 S-CoA S-CoA Isomers