Lecture 9: Pyrophin’s and Nucleotide Metabolism PDF

Pyrophin’s and Nucleotide Metabolism Porphyrin- A cyclic nitrogen containing compound that can bind to metal ions Fe 2+ and Fe 3+. Example- HEME ● Porphyrin Structure- 4 nitrogen containing pyrrole rings linked by methenyl bridges ● Heme- Prosthetic iron porphyrin, also known as Ferroprotroporphyrin...

Pyrophin’s and Nucleotide Metabolism Porphyrin- A cyclic nitrogen containing compound that can bind to metal ions Fe 2+ and Fe 3+. Example- HEME ● Porphyrin Structure- 4 nitrogen containing pyrrole rings linked by methenyl bridges ● Heme- Prosthetic iron porphyrin, also known as Ferroprotroporphyrin - Prosthetics- Aids enzymes in their functions ● Heme proteins are synthesized and degraded rapidly- To remain constant when red blood cells and other heme proteins are lost ● Heme is found in: Hemoglobin/Myoglobin/Cytochromes/Peroxidase/Catalase/Nitric Oxide Synthase ● Heme Biosynthesis- In liver and bone marrow ➢ Sites: Mitochondria and Cytosol ➢ Precursors: Glycine and Succinyl CoA (TCA intermediate) ➢ First Step Regulates: Catalyzed by Aminolevulinic Acid (ALA- Synthase), found in the mitochondria in 2 isoforms and X chromosome linked ➢ Important Intermediates: Aminolevulinic Acid (5-ALA) and Porphyrinogens ➢ Steps of Heme Biosynthesis 1. Glycine and Succinyl CoA become Aminolevulinic Acid (ALA) by ALA Synthase - ALAS 1 inhibited by Heme and ALAS 2 inhibited by iron ★ ALA Synthase Leaves Mitochondria- Enters Cytosol 2. 2 Aminolevulinic Acid (5-ALA) condense to form Pophobilinogen(has pyrrole ring) by ALA Dehydratase ★ LEAD: Inhibits ALA Dehydratase 3. 4 Porphobilinogen (PBG) molecules become hydroxymethylbilane (a linear tetrapyrrole) by Porphobilinogen Deaminase 4. Hydroxymethylbilane cyclizes to formUroporphyrinogen 3 5. Uroporphyrinogen 3 becomes coproporphyrinogen 3 by uroporphyrinogen decarboxylase - Coproporphyrinogen 3 goes into mitochondria (Series of Decarboxylation and Oxidation reactions of side chains) 6. Coproporphyrinogen 3 → Protoporphyrin IX 7. Protoporphyrin IX becomes Heme by Ferrochelatase and Fe 2+ cofactor ★ LEAD: Inhibits Ferrochelatase 8. Heme exits mitochondria and associates with globin chains ➢ Lead inhibition of ALA dehydratase and Ferrochelatase causes ALA elevation and lead associated anemia ➢ Regulation of Heme Synthesis ➔ Heme abundant: Biosynthesis decreases Pyrophin’s and Nucleotide Metabolism - Two Ways: Heme represses the synthesis of ALA synthase by transcriptional regulation or inhibits the activity of ALA synthase ➔ Heme low: Biosynthesis increases ➢ Porphyrias- Deficnecy of enzyme in Heme synthesis - Autosomal dominant or Autosomal Recessive - Accumulation of Porphyrin Intermediates- Excretion of intermediates in urine and feces - Features when enzyme defect is upstream of tetrapyrroles- Abdominal pain/Psychiatric manifestations/Neurologic manifestations - Types - Classified and disease manifestation depend on location of enzyme deficiency 1. Hepatic 2. Erythropoietic 3. Both Hepatic and Erythropoietic - Porphyria Cutanea Tarda- Occurs on sun exposed hands, photosensitivity (complement/mast cell) Pyrophin’s and Nucleotide Metabolism ● Hemoglobin- Globular protein, tetramer with 4 polypeptide chains ➢ Adult Hemoglobin (HbA1)- 2 alpha + 2 beta chains ➢ Each of the 4 polypeptide chains has one heme associated ➢ Heme of Hemoglobin- Contains Fe2+, ferrous in center - Iron bound to nitrogens of porphyrin ring and to a histidine residue of the polypeptide chain - Iron can form bonds with O2 ➢ Tertiary structure of globin polypeptide chains - Heme is in a hydrophobic pocket- To protect in from the aqueous environment - Alpha globin/Beta globin/Myoglobin have similar secondary and tertiary structures ➢ Quartanery Hb structure- Deoxy vs Oxy Hemoglobin - Oxy Hb (R-State)- Relaxed ★ Non-Covalent interactions between heterodimers are weaker ★ Cavity between beta subunits is smaller- 2,3 bisphosphoglycerate (2,3 BPG) released - Deoxy Hb (T-State)- Tense/Tight Pyrophin’s and Nucleotide Metabolism ★ Non-Covalent interactions between heterodimers are stronger ★ Cavity between the beta subunits is larger- Bridged by 2,3 bisphosphoglycerate (2,3 BPG) ★ Alpha and beta dimers are connected by salt bridges (ionic interaction) - 2,3 bisphosphoglycerate (2,3 BPG) ★ 2,3 bisphosphoglycerate (2,3 BPG) RBC concentration is equal to Hb concentration ★ Increase in 2,3 BPG stabilize T-state of Hb by cross linking B subunits through salt bridges ★ Increase in 2,3 BPG promotes oxygen release ★ Increase in 2,3 BPG occurs in response to tissue hypoxia ➢ Myoglobin- Oxygen storage protein - Location is in the cytosol of skeletal/cardiac/smooth muscle cells - Binds oxygen that was released by hemoglobin in tissue capillaries and diffused to tissue - Stored oxygen is available to the mitochondria ● Oxygen- Dissociation Curve: Oxygen saturation vs PO2, Sigmoid curve ➢ Displays how effectively Hb released O2 in tissues ➢ Increase in affinity- The binding of oxygen to 1 polypeptide chain of hemoglobin increases the binding of another oxygen molecules to the 2nd polypeptide chain ➢ Affinity Can be Shifted (Allosteric Effectors of Hb) - Right Shift- Decrease O2 affinity, more O2 released ★ Increase in PCO2- Co2 can bind to deoxy Hb, carbamino-Hb ★ pH decrease- H+ can bind to deoxy Hb ★ Increase in 2,3 BPG- It can bind to Hb ★ Above binding sites are different from O2 binding sites - Left Shift- Increased O2 affinity, less O2 released ★ Decrease in PCO2★ pH increase ★ Decrease in 2,3 BPG ★ Above binding sites are different from O2 binding sites ➢ Carbon Monoxide (CO)- Has a greater affinity fHemoor Hb than O2, it can bind to the iron of Heme - Smokers have higher levels of Carboxyhemoglobin (COHb) ➢ O2 bindings changes Hb from deoxy confirmation to oxy conformation ➢ Conditions - Hb is 50% saturated when PO2 is 26 mm Hg Pyrophin’s and Nucleotide Metabolism - ● ● ● ● Hb is 95% saturated when PO2 is 100 mm Hg (PO2 of arterial blood/leaving lungs) - Hb is 75% saturated in resting muscle capillaries when PO2 is 45 mm Hg ➢ Fetal Hemoglobin (HbF)- Higher affinity for O2, allowing for exchange of O2 between mother and fetus - HbF binds to 2,3 BPG less efficiently, so O2 curve is shifted towards left - After birth: Gamma gene is turned off and Beta gene is turned on - After 6 months: Most Hb is HbA1 (adult form) HbA1c-Glycosylated hemoglobin, glucose is covalently bound to N terminus of beta chains ➢ 5% is normal usually ➢ Diabetes- HbA1C can be as high as 12% Hemoglobinopathies- Genetic disorders, where there is abnormal Hb ➢ Sickle cell: Structurally abnormal Hb, HbS - Autosomal recessive, chronic hemolytic disease - Signs- Hemolytic anemia and recurrent pain ★ Vaso-occlusive- Causes pain in chest/bones/abdomen ➔ Misshapen sickle red blood cells become trapped in small capillaries and block circulation - Sickle cell disease (Sickle cell anemia)- Homozygous, two copies of mutant beta gene - Sickle cell trait- Heterozygous, one copy of mutant beta gene ★ Variant of beta-globin gene called sickle hemoglobin (Hb S) - Precipitating factors (Triggers)- Low O2/ High PCO2/ Low pH/ Concentration of sickle hemoglobin ➢ Thalassemias: Insufficient amount of Hb - Imbalance in synthesis of one of the two globin chains - Anemia: Can be caused by abnormal or non-functioning globin genes (insufficient synthesis) - Beta Thalassemia- Decreased synthesis of Beta globin chains - Homozygous B-Thalassemia- Cooley’s Anemia ➢ Other Hemoglobinopathies - Abnormal solubility of HbS- Hemolytic anemia and Pain - Ferric Heme- Methemoglobin (HbM)- Cyanosis and Hypoxia - Abnormal globin synthesis- Thalassemia- Anemia Cytochrome P450- Hemoprotein of Liver ➢ Detoxification and excretion of drugs/foreign substances ➢ Some drugs- Can induce enzymes of hepatic-metabolizing systems that have P450 Heme Degradation Pyrophin’s and Nucleotide Metabolism ➢ RBC’s are turned over after 4 months ➢ First step: Formation of bilirubin within macrophages of reticuloendothelial system ➢ Bilirubin is delivered to the the liver bound to plasma albumin ➢ Heme→ Biliverdin→Bilirubin - Iron is heme is recycled not excreted ➢ Hepatocytes: Bilirubin conjugated, called Bilirubin di-glucuronide - Conjugated bilirubin transported through the bile ducts of the liver and into the bile ➢ Intestines: Some conjugated bilirubin is converted to urobilinogen - Urobilinogen can be converted to stercobilin: Gives feces its brown colors - Some Urobilonogen is absorbed into portal circulation while some travels back to liver and is reabsorbed into bile ➢ Kidneys: Other urobilinogen is converted to urobilin and excreted by urine ➢ Hyperbilirubinemia - Normal Serum Bilirubin- Less than 0.8 mg/dl, most is unconjugated - In hyperbilirubinemia, level is greater than 2.5 mg/dl, leads to jaundice (icterus) Pyrophin’s and Nucleotide Metabolism ➢ Jaundice- Skin and eyes yellow, deposition of bilirubin from increased bilirubin levels in the blood - Cases with massive hemolysis: Liver is unable to conjugate the heme fast enough, which causes high unconjugated bilirubin in the bloodstream - Damage to liver cells: Unconjugated bilirubin levels in the blood rise - Inefficiency in conjugation activity - Bilirubin Entering Liver By Portal Circulation: Not reabsorbed into bile, goes to the kidney and darkens the urine, this in turn makes stool pale - Bile Ducts-Obstruction: Bilirubin will no pass to the intestine, yielding a pale stool and dark urine because conjugated bilirubin is delivered from the bloodstream - Causes 1. Prehepatic- Hemolysis/Autoimmune diseases/Abnormal hemoglobin 2. Intrahepatic- Infection/Drugs/Neonatal (bilirubin uridine diphosphate glucuronosyltransferase) 3. Posthepatic- Intrahepatic bile duct obstruction/Extrahepatic bile duct obstruction/Gallstones Nucleotide Metabolism ● Nucleotides- Phosphate esters of pentose sugar ➢ Base is linked to carbon one of the sugar ➢ Nucleotide triphosphates are precursors of nucleic acid (monomer) ➢ Functions - Energy compounds (ATP) - Co factors (NAD+/FAD+) - Not involved in metabolism ➢ Structure- Three components ➔ Nitrogenous Base/ 5 carbon sugar/ Phosphate ➢ Two Heterocyclic ➔ Purine- 2 rings - Guanine/Adenine ➔ Pyrimidines- 1 ring - Thymine/Cytosine/Uracil ➢ Nucleosides- Nitrogenous base + 5 carbon sugar ➢ Nucleotide- Phosphorylated Nucleoside ➢ Nucleotides come from - De novo synthesis of inosine monophosphate (IMP)- Which is a purine derivative with hypoxanthine (base) - Diet- Preformed nucleotides - Salvage Pathways- Recycling of endogenous nucleic acids Pyrophin’s and Nucleotide Metabolism ● Nucleotide Synthesis ➢ Purine and Pyrimidines synthesized de novo from amino acid and carbohydrate precursors ➢ Involve- Amino acids/ Phosphoribosylpyrophosphate (PRPP, Activated Ribose)/Tetrahydrofolate (FH4) ● Purine Synthesis- De Novo ➢ Identical In: E. Coli/Yeast/Pigeons/Humans ➢ Steps 1. Ribose 5 Phosphate becomes phosphoribosylpyrophosphate (PRPP) by PRPP synthase 2. PRPP is animated: A phosphate is removed and replaced with an amine group - PRPP is also a precursor for pyrimidine biosynthesis 3. Multiple phosphorylation occur by ATP: Carbonyl groups are displaced with amine groups, Inosine Monophosphate formed - Atom sources: Glycine/Glutamine/Aspartic Acid/THF-formyls/CO2 4. Ionosine Monosphate (IMP) is aminated to yield Adenosine Monophosphate (AMP) and Guanosine Monophosphate (GMP), which are ribose sugars - IMP is a precursor for pyridine synthesis, it is rapidly converted - Nucleoside monophosphate/diphosphate kinasesPhosphorylation 5. AMP and GMP can be converted to ATP and GTP by phosphorylation by kinases ➢ Purine Nucleotide Synthesis - IMP is a precursor for pyridine synthesis, it is rapidly converted in nucleotide synthesis - Nucleoside monophosphate/diphosphate kinases- Phosphorylation - Adenylate Kinase- Forms ADP - Guanylate Kinase- Catalyzes phosphorylation of GMP to GDP ➢ Regulation of Purine Biosynthesis- At first two enzymatic reactions - Ribose Phosphate Pyrophosphokinase- Catalyzes transformation of Ribose 5 phosphate to PRPP - Amindo Phosphoribosyl Transferase- Catalyzes transformation of PRPP into B-5-Phosphoribosylamine (PRA) - Regulation- IMP production controlled by concentrations of both adenine nucleotides and guanine nucleotides ➔ Feedforward- Increasing ➔ Inhibition- Decreasing Pyrophin’s and Nucleotide Metabolism - GTP provides energy for AMP synthesis while ATP provides energy for GMP synthesis ➢ Purine Nucleotide Degradation - Purine nucleotides are degraded to uric acid and excreted in the urine - Hypoxanthine (base) is oxidized to uric acid by xanthine oxidase - Hypoxanthine can be utilized by the salvage pathway- Reacts with activated ribose to form IMP nucleotide ➔ Enzyme involved in salvage- Hypoxanthine guanine phosphoribosyl transferase (HGPRT) - Salvage Pathway ➔ Adenine: Adenine + PRPP = AMP + Phosphate - Adenine phosphoribosyltransferase (ARPT) ➔ Hypoxanthine + PRPP = IMP+ phosphate/ Guanine + PRPP = GMP+ phosphate - Hypoxanthine guanine phosphoribosyl transferase (HGPRT) - LESCH-NYHAN Syndrome- Caused by HGPRT deficiency ★ Sex linked congenital defect in mostly males ★ Excess uric acid production- Product of purine degradation ★ Deficiency Hypoxanthine guanine phosphoribosyl transferase (HGPRT)- Inability to salvage hypoxanthine and guanine ★ System will have excess PRPP which activates amidophosphoribosyltransferase creating more IMP and GMP - Purine amount increases (and ultimately uric acid) ★ Neurological abnormalities resultsSpasticity/Mental deficits/Aggressive and destructive behavior including self harm ➢ Purine Metabolism- Gout - Hyperuricemia- Urate crystals are deposited in cartilage, joints, and kidneys - Recurrent attacks of acute arthritis - Can be genetic, may be due to overproduction of uric acid, excessive purine nucleotide synthesis, or defective renal excretion of uric acid - Gout can be secondary to other diseases - Treatments Pyrophin’s and Nucleotide Metabolism ➔ Allopurinol (Zyloprim)- Hypoxanthine analog, Inhibits xanthine oxidase to lower uric acid production ➔ Colchicine- Antiflmmatory for acute arthritic attacks ➢ Adenosine Deaminase Deficiency - Adenosine Deaminase- Enzyme of purine nucleotide degradation - Autosomal recessive deficiency - Deficiency causes severe combined immunodeficiency (SCID) - Lack of T and B cells- Hard to fight infection - Mechanism ➔ Deoxyadenosine is phosphorylated to form high amount of dATP ➔ High dATP concentration inhibits ribonucleotide reductase ➔ RESULTS- Synthesis of other dNTP’s is reduced/DNA synthesis inhibited/ Cell Proliferation decreases/SCIDS ● Pyrimidine Synthesis ➢ Regulation- Carbamoyl phosphate synthesized from bicarbonate and Glutamine-derived ammonia by Carbamoyl phosphate synthetase ➢ Orotate ring is formed from carbamoyl phosphate and aspartate ➢ Coupling occurs to activate ribose-PRPP to form orotidylate- Orotidine 5 monophosphate (OMP) ➢ IMPORTANT- OMP is decarboxylated to form uridine (UMP) ➢ Phosphates are transferred to UMP to form UTP ➢ UTP amination to form CTP ➢ Generation of UMP Involves Enzymes - E1- Regulation- Carbamoyl phosphate synthetase - E2- Aspartate transcarbamylase (ATCase) - E3-Dihydroorotase - E4-Dihydroorotate dehydrogenase (mitochondria) - E5-Orotate phosphoribosyltransferase - E6- Orotidine-5’ monophosphate decarboxylase (OMP) - PRODUCES-UMP ➢ Pyrimidine Nucleotide Synthesis- UMP to UTP, similar to synthesis of purine triphosphates - UMP + ATP → UDP + ADP - UDP + ATP → UTP + ATP - CTP synthease drives these reactions to generate CTP - Methylenetetrahydrofolate (Thymidylate synthase)- Forms Thymidylate, thymine which is a pyrimidine base - Deoxythymidine triphosphate (dTTP) in DNA synthesis is from Deoxyuridine monophosphate (dUMP) - Deoxyuridine diphosphate (dUDp) is hydrolyzed to dUMP, Pyrophin’s and Nucleotide Metabolism - Deoxyuridine monophosphate (dUMP) is converted to deoxythymidine monophosphate (dTMP): Methylation of deoxyribose from urdine, in a pathway that involves folates - Thymidylate synthase enzymes- Transfers methyl group to Deoxyuridine monophosphate (dUMP) to generate deoxythymidine monophosphate (dTMP) ➢ Pyrimidine Metabolism - Uridine Monophosphate (UMP) is the precursor of all pyrimidine nucleotides - De Novo Pathways- Ends with synthesis of UMP - Other pathways- Lead to the formation of cytidine triphosphate (CTP) and Thymidine Triphosphate (TTP) ➔ CTP is formed when UTP is aminated by CTP synthetase - Salvage pathways- Allow cells to reuse pyrimidines, similar to purines ➢ Formation of Deoxynucleotides- Deoxyribonucleotides of Adenine/Guanine/Cytosine synthesizes from ribonucleotides by reduction of 2-hydroxyl by ribonucleotide reductase - Nucleotides- Amino acid precursors and Phosphoribosylpyrophosphate - Salvage pathways - Ribonucleotide products are converted to deoxyribonucleotide by ribonucleotide reductase