Week 3 - Tetrahydrofolate, Vitamin B12, and S-Adenosylmethionine Flashcards
Describe how dietary folate and cobalamin are taken up from the diet and converted to active cofactors. Describe their different forms, and the types of reactions in which they participate, and the molecular basis for pathologies associated with their dietary or functional deficiency.
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Describe the molecular pathophysiology of: spina bifida, hyperhomocysteinemia, pernicious anemia, megaloblastic anemia, hereditary folate malabsorption, targeting folate metabolism for cancer treatment.
*key pathologies.. spinabifida - dont worry about mechanism..just pathology.
Describe the sources and recipients of one carbon transfer reactions. Describe how deficiency in cobalamin can affect other one carbon transfer reactions (the methyl trap hypothesis).
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*What are the oxidation states of carbon?
Reduced if has lots of H bound to it
What is outline of single carbon pool?
Refers to all different cofactors and substrates that bind cofactors to make single carbon transfers…FH4 is regenerated after carbon transfer..
What is first place carbons bind?
FH4
What is the vitamin precursor for FH4?
Folate
Where does folate come from in diet?
Green leafy vegetables, liver, legumes, yeast, and fortified flour
Folic acid deficiency causes?
Malnutrition or malabsorption… Need more folate during pregnancy
Spina bifida
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Why are women recommended to take folate during pregnancy?
Because folate deficiency before or during pregnancy is associated with neural tube defects such as spina bifida Take 400 micrograms daily a month before and three months after getting pregnant
What happens to poly-glutamate tail on folate? What is folate reduced to in intestinal epithelial cells? In liver?
It is digested in the gut down to mono-glutamate N5-methyl tetrahydrofolate Polyglutamated in liver
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Hereditary folate malabsorption
Inherited mutation in the proton coupled folate transporter (PCFT) Causes functional folate deficiency despite adequate folate in diet
How is FH4 produced from folate?
It is reduced to FH2 and again to FH4 by enzyme DHFR
What enzyme is used in Folate to FH4? What reducing agent?
DHFR - dihydrofolate reductase NADPH is reducing agent
What is active cofactor that can bind methyl groups?
FH4
After some reactions FH4 is turned back into FH2 and it needs to be reduced again back to FH4. What enzyme?
Dihydrofolate reductase
What is an important drug target in cancer? What drugs?
DHFR Methotrexate Trimethoprim Pyrimethamine
Methotrexate
Used in cancer (chemotherapy), rheumatoid arthritis
Trimethoprim
Antibacterial
Pyrimethamine
Antimalarial
FH4 to 5 methyl Fh4 reaction and intermediates
know the intermediate oxidation states.. 10 forymy., 5 10 methylene, 5 10 methyn, 5 methyl…
What are we trying to make from FH4
5-methyl THF
What substrates can be combined with FH4 to make 5-methyl THF?
Formate Histitdine Serine (glycine)
If amino acid can degrade into acetyl coa and acetoacetate it is ketogenic..
so tryptophan is.
Formate + FH4 makes… What can it be reduced to?
10 formyl THF and water (-NH4 in two steps) 5-methyl THF (3 reductions)
Histidine + FH4 makes… What is it reduced to?
5,10 Methenyl THF (-NH4 in two steps) 5-methylTHF (2 reductions)
Serine + FH4 makes.. What is it reduced to? Enzyme?
Serine Hydroxymethyl transferase and PLP Makes Glycine and 5,10 methylene THF then it is reduced to 5-methyl THF
What can 5 methyl THF make when combined with cobalamin?
Methylcobalamin
What is the important irreversible reaction in the reduction of FH4 when a cabin is added?
5,10 methylene THF to 5-methyl THF is IRREVERSIBLE - methyl trap
*What are the products and substrates of one carbon pool donations?
One carbon pool: Serine Glycine Histidine Formate Products of donations to FH4 thymidine nucleotide purine bases methionine and S-adenosyl methionine
IF have b 12 deficiency.. wont get regeneartion of THF
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What produces most singe carbons from?
Serine** most important single carbon donor
What is reaction from Serine to 5,10methylene FH4 and how is FH4 regenerated? What is the enzyme?
Oxidized carbon is donated from 5,10 methylene TH4 to dUMP to make TMP, FH2 is left To regenearate FH4, Fh2 is reduced back to FH4 by NADPH Enzyme Dihydrofolate reductase
What does thymidyulate synthase TS catalyze?
It reduces methylene carbon to methyl during its transfer to dUMP to makd dTMP. This leaves cofactor FH4 in the unreduced form, FH2
What does DHFR catalyze.
It reduces FH2 to FH4, regenerating the cofactor
*What does hyroxymethyltransferase do? Ask dr. s about this..
It will add a one carbon group from serine to FH4 to produce methylene FH4
What does cell need dTMP for?
It helps make a nucleotide base important in DNA synthesis
What is another symptom of folate deficiency
Spinibifida - due to lac of DNA synthesis
*Draw dUMP, dTMP FH4 loop reaction. Enzymes?
Thymidylate synthase (TS) reduces methylene carbon to methyl during transfer of dUMP to dTMP. FH2 is then reduced to FH4 by DHFR, then can accept anorther carbon group from serine to make glycine and dUMP (enzyme serine hydroxymethyltransferase)
What is megaloblastic anemia
Cells are not dividing …just growing due to slow DNA synthesis Cells cannot synthesize enough DNA to replicate their chromosomes… This is caused by the cell not able to produce dTMP These cells are most apparent in bone marrow but can also be seen in circulating blood
*What are two chemotherapy drugs? Problem with them?
Methotrexate 5-fluorouracil Cannot distinguish between human and cancer cells..thats why pts get bad side effects..
What does methotrexate block?
It inhibits dihydrofolate reductase (DHFR)
What does 5 - fluorouracil block?
It inhibits thymidylate synthase (TS)
What does drug methotrexate
Competitive inhibitor of dihydrofolate reductase (DHFR)
*Waht is SAM? What does it do?
S-adenosyl Methionine SAM provides the methyl groups for biosynthtic reactions and can regenerate homocysteine
*What can SAM donate a methyl group to? What does it form?
When reduced carbon is donated for biosynthetic rxns…. It is not donated form 5-methyl FH4 directly, it goes through SAM… The methyl group is first donated to homocystein to make methionine which then joins adenosine base to form S-adenosyl methionine (SAM) SAM provides the methyl groups for biosynthetic reactions and can regenerate homocystine
What is only reaction 5-methyl dihydrofolate can participate in?
Addition of cobalamin to form methylcobalamin
Methylcobalamin participates in only one reaction. what is it?
Donation of methyl group for homocysteine, to make methionine*
What is adenosylcobalamin (5’-deoxyadenosylcobalamin) involved in?
Catalyzing the reaction of the isomerization of a methyl group in converting methylmalonyl CoA to Succinyl CoA (branched chain fatty acid oxidation)*
What do R-binders do?
R-binders ar proteins secreted in gut to bind cobalamin, as they are digested they bind an intrinsic factor. This complex is taken up by intestinal epithelial cells and transported in blood as a complex with transcobalamin II protein
What does the intrinsic afactor do?
Bind to the R-binder cobalamin complex as they are digested
What is transcobalamin receptor
Takes in transcobalamin II protein
What is circulating form of cobalamin in blood?
Transcobalamin II protein
Where is cobalamin stored?
Liver In a complex called cubilin
What is cubillin?
The complex of cobalamin stored in the liver
What to parietal cells and gastric mucosa secrete? What if they dont work?
Parietal cells secrete the intrinsic factor Gastric mucosa secrete the R-binder
Symptoms of B12 deficiency?
Folate deficiency plus neurlogical issues….. Pernicious anemia (megaloblastic anemia plus neuropathy)
Where is vitamin B12 (cobalamin) found in diet?
Meat, eggs, and dairy (can be free or protein bound)
What is cobalamin reaction 1?
Methymalonyl CoA to Succinyl CoA
Catabolism of branched chain and odd chain length fatty acids and branched chain amino acids.. Cofactor for the reaction of Methylmalonyl CoA to Succinyl CoA, the linear substrate is a TCA cycle intermediate
Methylmalonyl CoA to Succinyl CoA using enzyme Methylmalonyl CoA mutase
Cobalin is in the form of Adenosyl cobalamin
Waht does adenosyl cobalamin do?
Cofactor for the enzyme that catalyzes the reaction from methylmalonyl CoA to Succinyl CoA.
Enzyme methylmalonyl CoA mutase
What is anaplerosis?
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What is the enzyme (cofactor vit B12) for reaction of methylmalonyl CoA to Succinyl CoA?
Methylmalonyl CoA Mutase
Cobalamin Reaction 2..
Homocystein to Methionine
Homocystine to Methionine catalyzed by methionine synthase… Transfer of methyl from methycobalamin to homocysteine to make methionine. Methylcobalamin is regenerated by accepting a methyl from fully reduced 5-methyl TH4
What is methionine synthase? What does a mutation in it cause?
Catalyazes reaction from homocysteine to methionine. Mutation causes hyperhomocysteinemia
What is hyperhomocysteinemia?
Arthorsclerotic plaques.. cardio issues.. still researching it… Caused by a mutation in methionine sythase
*Reaction of cobalamin (vit B12) and cysteine synthesis Name the cofactors for the reactions…
How is SAM made?
Methionine can bind an adenosine nucleoside to become S-adenosylmethionine (SAM). SAM donates methyl groups to numerous substrates, including precursors to neurotransmitters.
What 3 deficiencies can result in hyperhomocysteinemia?
B12 deficiency
Methionine synthase deficiency
Vitamin B6 deficiency
what is methyl trap hypothesis?
Only one fate for 5-methyl FH4… reacted with cobalamin to form methycobalamin IF cobalamin (vit B12) deficincy you get a methyl trap! All of TH4 is locked as 5 methyl hydrafolate.. Know reversible vs irreversible reactions.. 5-10 methyl to 5 methyl is one way trip*.. Now 5 methyl can only participate in is to maek methylcobalamin*** the only metabolic fate of 5 methyl THF.. If have vit b12 def you cant make cobalamin and therefore cannot make methylcobalamin.. 5 methyl THF builds up – this traps all of the folate in your body******** still have folate in blood if test for it though* That’s why B12 mimics folate def. B12 also has nerulogical symptoms: due to neruotransmitters.. .. Methylated product
what is methylmalonyl coa used for?
branched chain fatty acid..etc plus more
Case study
consanghinous parents (healthy(
what gene to look for?
gene markers are different in affected vs not affective children.. 1122 1122 etc.. Autosomal recessive (neither parent is affected) So could be PCFT (transporter name) .. SLC (gene name)… There are blood tests to look for folate. at first they were treated with oral folate.. but after they figured out the gene and transporter the children were injected with folate IM..
