lec 5 (mcbride) - one carbon metabolism

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43 Terms

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folic acid

  • folic acid (vitamin b9) = critical for reactions with one-carbon units

  • typical reaction type

    • transfer of one carbon components; thymine synthesis

  • consequences of deficiency

    • anemia

    • neural-tube defects in development

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sources of folic acid

  • naturally found in many foods

    • beans

    • peas

    • asparagus

    • eggs

    • leafy greens

    • beets

    • citrus fruits

    • brussel sprouts

    • broccoli

    • nuts and seeds

  • also added to foods and sold as supplement

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folic acid is made up of 3 components

  • pteridine ring

  • p-aminobenzoate (PABA)

  • glutamate

<ul><li><p>pteridine ring</p></li><li><p>p-aminobenzoate (PABA)</p></li><li><p>glutamate</p></li></ul><p></p>
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folic acid becomes…

polyglutamated

  • variable chain length polyglutamate tail serves to localize the molecule within cell

  • monoglutamates = actively transported across the cell membrane

  • for retention within the cell, folates are polyglutamates which redcues their affinity for the transporter and enhances their affinity for folate enzymes

<p>polyglutamated</p><ul><li><p>variable chain length polyglutamate tail serves to <u>localize the molecule within cell</u></p></li><li><p><strong>monoglutamates</strong> = actively transported across the cell membrane</p></li><li><p>for retention within the cell, folates are <strong>polyglutamates</strong> which<strong> redcues</strong> their affinity for the transporter and <strong>enhances</strong> their affinity for folate enzymes</p></li></ul><p></p>
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folic acid deficiency during pregnancy…

increases risk of neural tube defects

  • MRC vitamin study → showed that about 80% of neural tube defects could be prevented by taking 4 mg folic acid immediately BEFORE pregnancy

  • defects result when the neural tubes fail to close properly

    • rapidly dividing cells of the developing neural tube requires the synthesis of large amounts of nucleotides to facilitate DNA replication

<p><strong>increases</strong> risk of neural tube defects</p><ul><li><p>MRC vitamin study → showed that about 80% of neural tube defects could be prevented by taking <strong>4 mg folic acid</strong> <strong>immediately BEFORE pregnancy</strong></p></li><li><p>defects result when the neural tubes <u>fail to close properly</u></p><ul><li><p>rapidly dividing cells of the developing neural tube requires the synthesis of <u>large amounts of nucleotides</u> to facilitate DNA replication</p></li></ul></li></ul><p></p>
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folic acid must be converted to …

tetrahydrofolate (THF)

  • folic acid is sequentically reducedDHFTHF

  • enzyme dihydrofolate reductase (DHFR) carries out both steps

<p>tetrahydrofolate (THF)</p><ul><li><p>folic acid is sequentically <strong>reduced</strong> → <strong>DHF</strong> → <strong>THF</strong></p></li><li><p>enzyme <strong>dihydrofolate reductase (DHFR) </strong>carries out both steps</p></li></ul><p></p>
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tetrahydrofolate (THF)

is the coenzyme form

  • THF = important carrier of activated one carbon units

  • the one-carbon group carried by THF is bonded to its N-5 or N-10 nitrogen atom or to BOTH

<p>is the coenzyme form</p><ul><li><p>THF = important carrier of <strong>activated one carbon units</strong></p></li><li><p>the one-carbon group carried by THF is bonded to its <strong>N-5 or N-10</strong> <strong>nitrogen atom</strong> or to BOTH</p></li></ul><p></p>
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tetrahydrofolate is the carrier for one-carbon units in different oxidative states

one carbon groups carried by tetrahydrofolate

  • most reduced = methanol → -CH3 → methyl (group name)

  • intermediate = formaldehyde → -CH2- → methylene (group name)

  • most oxidized = formic acid → -CHO/-CHNH/-CH= → formyl/formimino/methenyl (group name)

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one carbon units attach to THF in an…

interconvertible manner

<p>interconvertible manner</p>
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folate cycle

the processing of modifying THF = folate cycle

  • 1C units are loaded onto THF and converted into usable forms for biosynthetic processes

  • folate cycle is compartmentalized and occurs in both the cytoplasm and mitochondria within cells

<p>the processing of modifying THF = folate cycle</p><ul><li><p>1C units are loaded onto THF and converted into usable forms for biosynthetic processes</p></li><li><p>folate cycle is <u>compartmentalized</u> and occurs in both the <u>cytoplasm</u> and <u>mitochondria</u> within cells</p></li></ul><p></p>
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THF is critical in…

de novo synthesis of glycine

  • de novo synthesis

    • glucose → serine → through THF glycine made

  • diet (protein)

    • creates both serines and glycine

<p>de novo synthesis of glycine</p><ul><li><p>de novo synthesis</p><ul><li><p>glucose → serine → through THF glycine made</p></li></ul></li><li><p>diet (protein)</p><ul><li><p>creates both serines and glycine</p></li></ul></li></ul><p></p>
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precursor of serine synthesis

3-phosphoglycerate is the precursor of serine synthesis

  • serine is synthesized from the glycolytic intermediate 3-phosphoglycerate

  • 3-phosphoglycerate is oxidized to 3-phosphohydroxypyrvuatetransaminated to 3-phosphoserinehydrolyzed to serine

<p><strong>3-phosphoglycerate</strong> is the precursor of serine synthesis</p><ul><li><p>serine is synthesized from the glycolytic intermediate 3-phosphoglycerate</p></li><li><p><strong>3-phosphoglycerate</strong> is <u>oxidized </u>to <strong>3-phosphohydroxypyrvuate</strong> → <u>transaminated</u> to <strong>3-phosphoserine</strong> → <u>hydrolyzed</u> to <strong>serine</strong></p></li></ul><p></p>
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precursor of glycine

serine is the precursor of glycine

  • in the formation of glycine, the side-chain methylene group of serine is transferred to THF

    • catalyzed by PLP enzyme serine hydroxymethyltransferase (SHMT1 or SHMT2)

<p>serine is the precursor of glycine</p><ul><li><p>in the formation of glycine, the <strong>side-chain methylene group of serine</strong> is transferred to THF</p><ul><li><p>catalyzed by <strong>PLP enzyme serine hydroxymethyltransferase (SHMT1 or SHMT2)</strong></p></li></ul></li></ul><p></p>
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experiment to understand the role of SHMT activity on circulating serine and glycine levels

LOK AT HIS VIDEO

<p>LOK AT HIS VIDEO</p>
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quantifying metabolite levels using liquid chromatography-mass spectrometry (LC-MS)

collection → extraction → injection → LC separation via HILIC column → ionization & detection by Q exactive plus MS instrument → MS spectrum → quantification

  • separate and detect up to 400 water soluble metabolites with a single 25 minute method

<p>collection → extraction → injection → LC separation via HILIC column → ionization &amp; detection by Q exactive plus MS instrument → MS spectrum → quantification</p><ul><li><p>separate and detect up to <strong>400</strong> water soluble metabolites with a single 25 minute method</p></li></ul><p></p>
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inhibiting SHMT 1/2 elevates glycine levels

  • SHIN inhibits SHMT1/2

  • SHMT1/2 converts serine → glycine while also transferring a 1C unit to THF; when inhibited, reaction is blocked, leading to buildup of glycine as in order for glycine → serine it needs the 1C-THF

<ul><li><p>SHIN inhibits SHMT1/2</p></li><li><p>SHMT1/2 converts serine → glycine while also transferring a 1C unit to THF; when inhibited, reaction is blocked, leading to <strong>buildup of glycine</strong> as in order for glycine → serine it needs the 1C-THF</p></li></ul><p></p>
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whole body glycine clearance

reverse SHMT flux in the liver is required for whole body glycine clearance

  • most tissues and proliferating cells have forward SHMT FLUX where serine → glycine

  • highlights the highly reservable nature of reactions with exchange of a 1C unit via the coenzyme THF

<p><strong>reverse SHMT flux</strong> in the liver is required for whole body glycine clearance</p><ul><li><p>most tissues and proliferating cells have <strong>forward SHMT FLUX</strong> where serine → glycine</p></li><li><p>highlights the highly reservable nature of reactions with exchange of a 1C unit via the coenzyme THF</p></li></ul><p></p>
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glycine is also cleared by glycine cleavage system

  • glycine cleavage system is a series of 4 enzymes that convert glycine → CO2 and 5,10-methyleneTHF

  • predominately active in liver

  • critical source of 1C units in early development

<ul><li><p>glycine cleavage system is a series of <strong>4 enzymes</strong> that convert <strong>glycine → CO2 and 5,10-methyleneTHF</strong></p></li><li><p>predominately active in <u>liver</u></p></li><li><p>critical source of 1C units in <u>early development</u></p></li></ul><p></p>
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SHMT and glycine cleavage system = major contributors to glycine clearance

  • historically, glycine cleavage system was thought to be major pathway of glycine clearance in mammals

  • however, liver knockout of either SHMT2 or major enzyme of the glycine cleavage system (GLDC) dramatically elevate glycine levels in mouse models

<ul><li><p>historically, glycine cleavage system was thought to be major pathway of glycine clearance in mammals</p></li><li><p>however, liver knockout of either SHMT2 or major enzyme of the glycine cleavage system (<strong>GLDC</strong>) dramatically <strong>elevate glycine levels</strong> in mouse models</p></li></ul><p></p>
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synthesis of methionine

1C unit, as 5-methylTHF, is required for the synthesis of methionine

<p>1C unit, as 5-methylTHF, is required for the synthesis of methionine</p>
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methionine cycle

is coupled to folate cycle

  • methionine = essential amino acid b/c the full carbon backbone CANNOT be synthesized so must be obtained from diet

<p>is coupled to folate cycle</p><ul><li><p>methionine = essential amino acid b/c the full carbon backbone CANNOT be synthesized so must be obtained from diet</p></li></ul><p></p>
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major donor of methyl groups

s-adenosylmethionine = major donor of methyl groups

  • SAM = activated methyl donor with higher transfer potential than THF

    • synthesized from methionine and ATP

<p><strong>s-adenosylmethionine</strong> = major donor of methyl groups</p><ul><li><p>SAM = activated methyl donor with <strong>higher transfer potential</strong> than <strong>THF</strong> </p><ul><li><p>synthesized from <u>methionine </u> and <u>ATP</u></p></li></ul></li></ul><p></p>
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methylation by SAM is…

critical modification for numerous biological functions

  • methylation of DNA, typically on cytosine ring, is generally repressive of gene expression

  • methylation of histone proteins, typically on lysine or arginine residues, can either promote or repress gene expression depending on the chromatin structure and recruitment of other proteins

  • methylation of RNA, typically on adenine, can influence splicing, stability, nuclear export, translation and other properties

  • methylation of proteins can play important regulatory roles by influencing structure

<p>critical modification for numerous biological functions</p><ul><li><p>methylation of DNA, typically on <u>cytosine ring</u>, is generally <strong>repressive of gene expression</strong></p></li><li><p>methylation of histone proteins, typically on <u>lysine</u> or <u>arginine</u> residues, can either <strong>promote or repress gene expression</strong> depending on the chromatin structure and recruitment of other proteins</p></li><li><p>methylation of RNA, typically on <u>adenine</u>, can influence splicing, stability, nuclear export, translation and other properties</p></li><li><p>methylation of proteins can play important regulatory roles by influencing structure</p></li></ul><p></p>
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SAM is converted to…

homocysteine

  • after donation of methyl group by SAM, resulting s-adenosylhomocysteine is hydrolyzed, yielding adenosine and homocysteine

<p><strong>homocysteine</strong></p><ul><li><p>after donation of methyl group by SAM, resulting <strong>s-adenosylhomocysteine</strong> is <strong>hydrolyzed</strong>, yielding <strong>adenosine</strong> and <strong>homocysteine</strong></p></li></ul><p></p>
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regeneration of methionine

  • methionine is regenerated by transfer of methyl group to homocysteine from N5-methyltetrahydrofolate

    • catalyzed by methionine synthase

    • mediated by the coenzyme methylcobalamin which is derived from vitamin B12

<ul><li><p>methionine is regenerated by <u>transfer of </u><strong><u>methyl group</u></strong><u> to </u><strong><u>homocysteine </u></strong><u>from</u><strong><u> N5-methyltetrahydrofolate</u></strong></p><ul><li><p>catalyzed by <strong>methionine synthase</strong></p></li><li><p>mediated by the coenzyme <strong>methylcobalamin</strong> which is derived from vitamin B12</p></li></ul></li></ul><p></p>
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deficiency of vitamin b12 or folic acid can lead to…

increased homocysteine levels

  • regularly homocysteine is converted back to methionine via methionine synthase which requires 5-methyl-THF (provides methyl curve), vit. B12 (acts as cofactor), methionine synthase (catalyzes the rxn)

  • WITHOUT folic acid, NO 5-methyl-THF so homocysteine CANNOT be converted into methionine

  • even if folate is present, B12 is required as cofactor and without it

<p><strong>increased homocysteine levels</strong></p><ul><li><p>regularly homocysteine is converted back to methionine via methionine synthase which requires 5-methyl-THF (provides methyl curve), vit. B12 (acts as cofactor), methionine synthase (catalyzes the rxn) </p></li><li><p>WITHOUT folic acid, NO 5-methyl-THF so homocysteine CANNOT be converted into methionine</p></li><li><p>even if folate is present, B12 is required as cofactor and without it </p></li></ul><p></p>
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high homocysteine levels…

correlate with vascular disease

  • elevated serum levels of homocysteine or the disulfide-linked dimer homocysteine = predisposing factor for coronary heart disease and arteriosclerosis

  • elevated homocysteine levels can also result from mutations in the gene encoding cystathionine β-synthase

  • high levels of homocysteine:

    • damage cells lining blood vessels

    • increase the growth of vascular smooth muscle

    • raise oxidative stress

    • implicated in the development of type 2 diabetes

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formation of SAM activates…

methyl group of methonine which the folate cycle restores

  • SAM loses active -CH3 → s-adenosylhomocysteine → hydrolyzed to homocysteine → gains -CH3 group (from folate cycle) to become methionine → ATP to become SAM

<p>methyl group of methonine which the folate cycle restores</p><ul><li><p>SAM loses active -CH3 → s-adenosylhomocysteine → hydrolyzed to homocysteine → gains -CH3 group (from folate cycle) to become methionine → ATP to become SAM</p></li></ul><p></p>
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nucleotides can be synthesized by de novo or salvage pathways

nucleotides are key biomolecules

  • activated precursors of nucleic acids

  • ATP = universal currency of energy

  • GTP = energy source

  • nucleotide derivatives participate in biosynthetic processes

  • cAMP and cGMP = essential components of signal-transduction pathways

  • ATP = acts as donor of phosphoryl groups transferred by protein kinases

de novo pathway

activate ribose (PRPP) + amino acids + ATP + CO2 + … → nucleotide

salvage pathway

activated (PRPP) + base → nucleotide

<p>nucleotides are key biomolecules</p><ul><li><p>activated precursors of nucleic acids</p></li><li><p>ATP = universal currency of energy</p></li><li><p>GTP = energy source</p></li><li><p>nucleotide derivatives participate in biosynthetic processes</p></li><li><p>cAMP and cGMP = essential components of signal-transduction pathways</p></li><li><p>ATP = acts as donor of phosphoryl groups transferred by <u>protein kinases</u></p></li></ul><p>de novo pathway</p><p>activate ribose (PRPP) + amino acids + ATP + CO2 + … → nucleotide</p><p>salvage pathway</p><p>activated (PRPP) + base → nucleotide</p><p></p>
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de novo and salvage pathways use different starting material for nucleotide synthesis

  • de novo pathways = pathways in which nucleobases are assembled from scratch

    • for pyrimidines, framework for the base is assembled first and then attached to ribose

    • for purines, base is synthesized piece by piece directly onto a ribose-based structure

  • salvage pathays = pathways in which preformed bases are recovered and reconnected to a ribose unit

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pyrimidine ring

assembled de novo from CO2, ammonia, aspartate

  • pyrimidine rings are assembled from bicarbonate, aspartate and ammonia

  • glutamate often serves as ammonia donor

<p>assembled de novo from <strong>CO2, ammonia, aspartate</strong></p><ul><li><p>pyrimidine rings are assembled from <strong>bicarbonate, aspartate and ammonia</strong></p></li><li><p><strong>glutamate</strong> often serves as <strong>ammonia donor</strong></p></li></ul><p></p>
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formation of PRPP

  • 5-phosphoribosyl-1-pyroposphate (PRPP) = form of ribose activated to accept nucleobases

    • synthesized by 5-phosphoribosyl-1-pyrophosphate synthetase

<ul><li><p>5-phosphoribosyl-1-pyroposphate (PRPP) = form of ribose activated to <strong>accept nucleobases</strong></p><ul><li><p>synthesized by <strong>5-phosphoribosyl-1-pyrophosphate synthetase</strong></p></li></ul></li></ul><p></p>
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glutamine and purine synthesis

glutamine provides the nitrogen via ammonia for the committed step in purine synthesis

  • purine bases are assembled already attached to the ribose ring

  • committed step in purine biosynthesis forms 5-phosphoribosyl-1-amien from PRPP and glutamine

    • catalyzed by glutamine phosphoribosyl amidotransferase

<p>glutamine provides the <strong>nitrogen</strong> via <strong>ammonia</strong> for the committed step in purine synthesis</p><ul><li><p><u>purine bases</u> are assembled already attached to the ribose ring</p></li><li><p>committed step in purine biosynthesis forms <strong>5-phosphoribosyl-1-amien</strong> from <strong>PRPP </strong>and <strong>glutamine</strong></p><ul><li><p>catalyzed by <strong>glutamine phosphoribosyl amidotransferase</strong></p></li></ul></li></ul><p></p>
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purine nucleotide synthesis

requires 1C units as 10-formylTHF from folate cycle

<p>requires 1C units as <strong>10-formylTHF</strong> from <u>folate cycle</u></p>
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de novo purine biosynthesis pathway

2 of the enzymes in the de novo purine biosynthesis pathway require 10-formyl-THF

glycinamide ribonucleotide → formylglycinamide ribonucleotide

3-aminoidazole-4-carboxyamide ribonucleotide → 5-formaminoimidazole-4-carboxyamide ribonucleotide

<p><strong>2 of the enzymes</strong> in the de novo purine biosynthesis pathway require <strong>10-formyl-THF</strong></p><p>glycinamide ribonucleotide → formylglycinamide ribonucleotide</p><p>3-aminoidazole-4-carboxyamide ribonucleotide → 5-formaminoimidazole-4-carboxyamide ribonucleotide</p>
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first chemotherapeutic agents

the first chemotherapeutic agents were antifolates

  • Dr. Sydney Farber treated 16 children with acute lymphoblastic leukaemia (AML) with aminopterin, amino derivative of folic acid

  • 10 of the pts → temporary remission

  • trial = first ever succesful remission of leukaemia and foundation of modern chemotherapy

  • aminopterin → developed to methotrexate; less toxic and commonly used in chemotherapy treatment

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methotrexate

is a folic acid derivative

  • chemotherapeutic agent in many types of cancer

  • immunosuppressant used to treat inflammatory conditions including rheumatoid arthritis

<p>is a folic acid derivative</p><ul><li><p>chemotherapeutic agent in many types of cancer</p></li><li><p>immunosuppressant used to treat inflammatory conditions including rheumatoid arthritis</p></li></ul><p></p>
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methotrexate is a DHFR inhibitor

  • inhibits dihydrofolate reductase (DHFR)

  • prevents generation of THF to carry 1C units for purine synthesis

<ul><li><p>inhibits dihydrofolate reductase (DHFR)</p></li><li><p>prevents generation of <strong>THF</strong> to carry 1C units for purine synthesis</p></li></ul><p></p>
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methotrexate and other antifolate therapies mechanism

block cell division by depleting 10-formylTHF levels and inhibiting purine synthesis

<p>block cell division by <strong>depleting 10-formylTHF levels </strong>and <strong>inhibiting purine synthesis</strong></p>
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in a patient with folic acid deficiency, what synthetic form of folic acid is optimal for this person to receive?

A. no glutamate tail

B. monoglutamated

C. di-glutamated

D. highly polyglutamated

answer = monoglutamated

  • polyglutamated would be stuck in circulation b/c it has poor affinity for transporter the tissues that need the folic acid won’t be able to take it up

  • monoglutamated lets it enter different tissues → polyglutamated to stay in cell

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how is 5,10-meTHF generated in the mitochondria transported to the cytoplasm for biosynthetic processes in the cellular compartment that require this form of a 1C unit?

A. passive diffusion

B. active transport

C. broken-down and re-synthesized

answer = broken down and re-synthesized

5,10-meTHF → 10-formylTHF via MTHFD2/L → formate via MTHFD1L → formate diffuses out of mitochondria and enters the cytoplasm → formate reconverted into 10-formylTHF via MTHFD1 → 5,10-meTHF via MTHFD1

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if a cancer cell line is growing in folic acid-supplemented culture media and then is suddenly switched into folic acid-free culture media, how would you predict the levels of serine and glycine would change?
A. serine increases and glycine decreases

B. serine decreases and glycine increases

C. serine and glycine both increase

D. serine and glycine both decrease

answer = serine increases and glycine decreases

  • removing vitamin precursor of folic acid → reduce THF → THF is substrate for SHMT rxn → less conversion of serine into glycine

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a patient consumes 100 mols of glycine supplement and all of the glycine is cleared in the liver; 25 moles is cleared by SHMT to make serine and 75 mols is cleared by glycine cleavage system to make CO2. what is the predicted change to 5,10-methyleneTHF levels in the liver?

A. increase by 10 mols

B. increase by 50 mols

C. decrease by 100 mols

D. decrease by 50 mols

answer = increase by 50 mols

  • through the SHMT rxn 25 mols of glycine is converted to serine which consumes 25 mols of 5,10-meTHF → THF

  • through glycine cleavage system, 75 mols of glycine make 75 mols of CO2 and also 5,10-meTHF

  • 75-25 = +50 mols