Biochemistry - Tetrahydrofolate, B12 and SAM 2023 PDF

Single Carbon Transfer Objective A • Some synthetic pathways require the addition/transfer of single carbon groups that exist in a variety of oxidation states • One-carbon groups at lower levels of oxidation than CO2 (-CHO, =CH-, -CH2,-CH3) are transferred from carrier compounds to specific struct...

Single Carbon Transfer Objective A • Some synthetic pathways require the addition/transfer of single carbon groups that exist in a variety of oxidation states • One-carbon groups at lower levels of oxidation than CO2 (-CHO, =CH-, -CH2,-CH3) are transferred from carrier compounds to specific structures that are being synthesized or modified • [CO2 is the most oxidized form of carbon] • Carrier compounds: • Tetrahydrofolate (FH4) ß derivative of vitamin B9 folate • Vitamin B12 (cobalamin) ß vitamin • S-adenosylmethionine (SAM) ß metabolic cofactor synthesized by the body. 3 Folate = Vitamin B9 à Tetrahydrofolate (FH4) • Tetrahydrofolate (FH4) is produced from the vitamin folate and is the primary one-carbon carrier in the body. • 3 major structural components: – Bicyclic pteridine ring – para-aminobenzoic acid (PABA) – Polyglutamate tail • The one-carbon group accepted and transferred is bound to N5, N10, or both. • Forms of folate may differ in the oxidation state of one-carbon group, number of glutamate residues attached, or degree of oxidation of pteridine ring. – Term “folate” (or synthetic “folic acid”) means the most oxidized form of pteridine ring – Folate is reduced to FH2 and then to FH4 by dihydrofolate reductase – Since reduction is the favored direction of the reaction; most folate is present in the reduced coenzyme form, FH4. Objective B 4 Folate – Vitamin B9 à Tetrahydrofolate • Folate is synthesized in bacteria and higher plants and ingested in green leafy vegetables, fruits, and legumes in the diet. • Most dietary folate derived from food is present in the reduced coenzyme form (FH4). • However, vitamin supplements and fortified food contain the oxidized form of the pteridine ring. • After ingestion, the glutamate residues of dietary folate are cleaved off in the intestine to produce the monoglutamate form of folate which is then absorbed. • Within the intestinal cells, folate is converted principally to N5-methyl-FH4 which is then carried to the liver. • The liver (which stores ½ of the body’s folate) reconjugates FH4 to the polyglutamate form before it is used in reactions. • N5-methyl-FH4 is the major form in the blood. 5 Oxidation and Reduction of the One-Carbon Groups of FH4 • One-carbon groups are attached to either N5, N10, or form a bridge between both. • The collection of one-carbon groups attached to FH4 is known as the one-carbon pool. • While attached to FH4, the one-carbon units can be oxidized or reduced. • Most oxidized form of carbon is N10-formyl-FH4 • Most reduced form of carbon is N5-methyl-FH4 • Once methyl group is formed, it is not readily reoxidized back to N5, N10-methylene-FH4, thus N5-methyl-FH4 tends to accumulate in the cell. Objective B 6 Sources and Recipients of One-Carbon Groups Carried by FH4 • Carbon sources for the one-carbon pool include: • Serine (*major carbon source in humans) • Glycine • Formaldehyde • Histidine • Formate • Precursors Products • Serine Glycine • Deoxyuridine monophosphate (dUMP) dTMP • Purine base precursor C2 and C8 of purine ring • Vitamin B12 B12– CH3 Objective C 7 FH4 and dTMP Synthesis Objective D • FH4 is required for the synthesis of deoxythymidine monophosphate (dTMP) needed for DNA synthesis. Therefore, FH4 is required for optimal cell growth and cell division, which can be restricted by a dietary deficiency of folate. • Note the methylene form of FH4 is used in the conversion of dUMP to dTMP, yet it is a methyl group that is transferred in the reaction. FH4 donates two electrons to the methylene group to reduce it to a methyl group and form the single bond to the carbon atom, resulting in oxidized dihydrofolate (FH2). • FH2 must be reduced to FH4 to regenerate the active form of the cofactor. This is done by dihydrofolate reductase, using electrons provided by NADPH. The FH4 is reloaded with a –CH2- group from a serine. 9 Anti-Cancer Drugs that Interrupt dUMP à dTMP 5-Fluorouracil is a structural analog of dTMP that has high affinity for the tymidylate synthase active site. Acts as a competitive inhibitor for dUMP and methylene FH4 binding. Blocks production of dTMP and greatly restricts DNA synthesis in rapidly dividing cells. Methotrexate is a structural analog of folate that is a competitive inhibitor of dihydrofolate reductase, preventing regeneration of fully reduced FH4 needed for the conversion of dUMP to dTMP 5-Fluorouracil Folate Methotrexate Objective D 10 Folate Deficiency and Fetal Neural Tube Development • Folate deficiency in pregnant women, especially during the month before conception and the month after, increases the risk of neural tube defects anencephaly and spina bifida in the fetus. • To reduce the risk, women in child-bearing age, are recommended to take 400 µg of folic acid in a multivitamin 11 Folate Deficiency • Folate deficiencies occur frequently in chronic alcoholics • Megaloblastic anemia is a characteristic of folate deficiency • Several factors are involved: • Aspirate usually contains a greater than normal # of RBC and WBC precursors, most of which are larger than normal • Inadequate dietary intake of folate • Direct damage to intestinal cells and brush border enzymes (interferes with absorption of dietary folate) • A defect in the enterohepatic circulation, which reduces the absorption of folate • Liver damage that causes decreased hepatic production of plasma proteins • Interference with kidney reabsorption of folate • There is a risk for a direct toxic alcohol effect on hematopoietic tissues \bone marrow aspirates are usually taken • These large RBC precursors are called megaloblasts. • Hematopoietic precursor cells exposed to too little folate and/or vitamin B12 show slowed cell division, but cytoplasmic development occurs at a normal rate • Hence, megaloblasts tend to be large and have an increased ratio of RNA to DNA • Production of RBCs is decreased, a condition called ineffective erythropoiesis 12 Sulfa Drugs Objective E Antibacterial sulfonamides (also called Sulfa drugs) are synthetic antimicrobial agents that contain the sulfonamide group. • Sulfa drugs are used in the treatment of certain bacterial infections WHY? • Unlike humans, bacteria must synthesize folate (not absorbed through dietary intake) • p-aminobenzoic acid (PABA) is an integral intermediate in the bacterial synthesis of folate • Sulfa drugs are analogs of PABA and act as a competitive inhibitor of the conversion of PABA to folate by dihydropteroate synthetase (DHPS). • Therefore, sulfa drugs prevent growth and cell division in bacteria • Because humans cannot synthesize folate, sulfa drugs do not affect human cells 13 Vitamin B12 (Cobalamin) Objective F • Complex structure • Corrin ring coordinated with cobalt (similar to heme-porphyrin ring coordinated with iron) • In the body it reacts with carbon of: • X: CH3 = methylcobalamin • X: 5’-deoxyadenosine = 5’deoxyadenosylcobalamin • X: CN = cyanocobalamin (commercial form) 14 Absorption and Transport of Vitamin B12 Objective F • Ingested B12 can exist in 2 forms: free or bound • If free, B12 binds to haptocorrins (R-binders, also known as transcobalamin I) secreted by salivary glands or gastric mucosa • If bound, digestive proteases in stomach release B12 to bind with R-binders • In small intestine, pancreatic proteases digest R-binders and the released B12 binds to an intrinsic factor • Intrinsic factor-B12 complex attaches to specific receptors in the ileum and is internalized • B12 complexes with carrier protein transcobalamin II and is released in circulation • Transcobalamin II-B12 complex delivers B12 to tissues (50% to liver) Objective f 15 Functions of Vitamin B12 Objective G • Vitamin B12 is involved in 2 reactions in the body A. Transfer of a methyl group from N5-methyl-FH4 to homocysteine to form methionine B. Rearrangement of L-methylmalonyl-CoA to form succinyl-CoA Reaction A: Transfer of –CH3 from FH4 to homocysteine to form methionine • FH4 receives a one-carbon group (typically from FH4) that is reduced to the methyl level and transferred to B12 forming methylcobalamin. • Methylcobalamin transfers the methyl group to homocysteine which is converted to methionine Objective g 16 Functions of Vitamin B12 Objective G Reaction B: Conversion of L-methylmalonyl-CoA to succinylCoA (Methylmalonyl CoA Mutase) • Active form of coenzyme is 5’-deoxyadenosylcobalamin • Part of metabolic route for the conversion of carbons from valine, isoleucine, threonine, and the last three carbons of odd-chain fatty acids, all of which form propionyl-CoA, to the TCA cycle intermediate succinylCoA. Objective g 17 Vitamin B12 Deficiency Objective H Very often Vitamin B12 and FH4 work together on methyl transfer reactions FH4 Methyl-FH4 Most notable changes due to B12 deficiency are chronically low levels of products dependent on Vitamin B12 methyl group transfer nearly all FH4 will accumulate in the methyl-FH4 form. 18 The Methyl Trap Hypothesis Objective H • The appearance of a functional folate deficiency caused by a lack of vitamin B12 is known as the methyl-trap hypothesis. • In B12 deficiency, the utilization of the N5-methyl-FH4 in the B12-dependent methylation of homocysteine to methionine is impaired. • Because N5-methyl-FH4 cannot be oxidized to any other FH4 forms, folate is now “trapped” in the N5-methyl form which accumulates. • The levels of other folate states decrease • THUS, B12 deficiency leads to folate deficiency FH4 Methyl-FH4 Decrease in N5, N10-methylene-FH4 and N10-formyl-FH4 needed for the synthesis of nucleotides required for DNA replication à megaloblastic anemia 19 Vitamin B12 Deficiency • Hematopoetic - caused by adverse effects on folate metabolism. Megaloblastic anemia (similar to folate deficiency). Lack of B12 causes folate deficiency by trapping FH4 as methyl-FH4. No free FH4 available for dTMP synthesis. • Neurological – progressive demyelination caused by inability to convert methylmalonyl-CoA to succinyl-CoA in the brain. Methylmalonyl-CoA accumulation interferes with myelin formation. 20 S-adenosylmethionine (SAM) • Metabolic cofactor that humans can synthesize. • SAM is used in reactions that add methyl groups to oxygen or nitrogen • More than 35 reactions in humans require SAM • SAM is synthesized from methionine and ATP (donates adenosine) • After methyl transfer to precursor, SAM forms S-adenosylhomocysteine (SAH) which is hydrolyzed to form homocysteine and adenosine • Homocysteine can accept a methyl group from methyl-B12 to regenerate Methionine Objective I 21 Single Carbon Transfer – Big Picture 22 Sample Question The figure below shows tetrahydrofolate (FH4) without or with single carbon groups attached. Which of the choices depicts FH4 with a carbon bound that is in the most oxidized state? A B C D E A B C D 23 Sample Question What is the role of tetrahydrofolate (FH4) in the conversion of dUMP to dTMP? A. FH4 provides a methyl group to dUMP to convert it to dTMP. B. FH4 provides a –CH2- group and two electrons to convert dUMP to dTMP. C. FH4 reacts with 5-fluorouracil to produce dTMP. D. FH4 reduces methotrexate so it can pass electrons to the dUMP molecule. E. FH4 removes the oxygen atom at the 2’ position on the TMP ribose ring. 24 Sample Question During a routine blood test, a patient had a very high level of methyltetrahydrofolate in her blood and almost no other form of tetrahydrofolate in the blood. What is the most likely cause of this finding? A. Dihiydrofolate reductase deficiency. B. Lack of adequate NADPH for the reactions. C. Vitamin B12 deficiency. D. S-adenosyl methionine deficiency. E. Tetrahydrofolate deficiency 25 Thank You! 26