4/8 one carbon metabolism

• FOLIC ACID

  • coenzyme: tetrahydrofolate
  • typical reactions type: thymine synthesis
  • consequences of deficiency: anemia, neural tube defects in development

  folate = critical for 1c unit rxns
• sources of folic acid

  • naturally many foods: beans, peas, asparagus, eggs, leafy greens, beets, citrus fruits, Brussel sprouts, broccoli, nuts and seeds
  • It is also added to foods and sold as a supplement
• FOLIC ACID IS MADE UP OF WHAT 3 COMPONENTS?

  - PTERIDINE RING, P AMINO BENZOATE (PABA), GLUTAMATE
• FOLIC ACID BECOMES POLYGLUTAMATED

  • reduces their affinity for transporter
  • enhances affinity for folate enzymes
• if u r pregnant and have a folic acid deficiency....THIS INCREASES RISK OF NEURAL TUBE DEFECTS
• TETRAHYDROFOLATE (coenzyme form)

  • carrier for one carbon (1c) units in different oxidative states
  • FOLIC ACID SEQUENTIALLY REDUCED TO FIRST DIHYDROFOLATE (DHF) AND THEN THF
  • **folic acid has to be converted to tetrahydrofolate (THF)
  • how? via enzyme DHFR (DIHYDROFOLATE REDUCTASE)
• table:

  most reduced= methanol

  intermediate = formaldehyde

  most oxidized = formic acid
• one carbon units attach to tetrahydrofolate in an interconvertible manner . n5 or n10
• what is the process of modifying tetrahydrofolate called?

  folate cycle

  • compartmentalized
  • occurs in BOTH cytoplasm and mitochondria within cells
  • One carbon units are loaded onto THF and converted into usable forms for biosynthetic processes
• tetrahydrofolate is critical in de novo synthesis of glycine
• Serine is the precursor of glycine

  • In the formation of glycine, the side-chain methylene group of serine is transferred to tetrahydrofolate.

  – catalyzed by the PLP enzyme serine hydroxymethyltransferase (SHMT1 or SHMT2)
• 3-phosphoglycerate is the precursor of serine synthesis

  • Serine is synthesized from the glycolytic intermediate 3-phosphoglycerate.

  • 3-Phosphoglycerate is oxidized to 3-phosphohydroxypyruvate, transaminated to 3-phosphoserine, and hydrolyzed to serine.
• liquid chromatography- mass spectrometry (LC-MS)

  1. collection
  2. extraction
  3. injection
  4. LC separation via HILIC column
  5. ionization and detection by Q EXACTIVE PLUS MS INSTRUMENT
  6. MS SPECTRUM
  7. quantification
• inhibiting SHMT1/2 elevates glycine levels

  • as glycine levels go down, serine levels go up

  
  

  serine is precursor of glycine. side chain methylene group of serine is transferred to tetrahydrofolate... catalyzed by SHMT1 (serine hydroxymethyltransferase)
• REVERSE SHMT flux is required for whole body glycine clearance.

  • if SHMT1/2 blocked, consumption decreases...production increases ... glycine levels increases bc it is not being cleared

  
  

  reaction: SERINE ---SHMT1/2---> GLYCINE

  SERINE <-------------GLYCINE

  
  

  REVERSIBLE REACTION :

  If SHMT is blocked: S CANT CONVERT TO G MEANING LESS G AND MORE S
• glycine is also cleared by glycine cleavage system

  • series of 4 enzymes that converts glycine --> co2 and 5,10-me THF
  • active in liver
  • critical source of 1-C units in early development
• both SHMT and glycine cleavage system are major contributors to glycine clearance.

  • glycine cleavage system was thought to be the major pathway of glycine clearance in mammals
  • SHMT2 or a major enzyme of the glycine cleavage system ELEVATE glycine levels in mouse models
• A one-carbon unit is required for the synthesis of methionine
• Methionine cycle is coupled to folate cycle.
• methionine is an essential amino acid bc the full carbon backbone cant be synthesized so needs to be obtained from diet
• S-Adenosylmethionine is the major donor of methyl groups

  • S-adenosylmethionine (SAM) = an activated methyl donor with higher transfer potential than tetrahydrofolate

  – synthesized from methionine and ATP
• Methylation by SAM is a critical modification for numerous biological functions.

  • generally repressive of gene expression
  • either promote or repress gene expression depending on the chromatin structure and recruitment of other proteins (i.e. transcription factors)
  • methylation of RNA, typically on adenine, can influence splicing, stability, nuclear export, translation, and other properties
  • methylation proteins can play important regulatory roles by influencing structure
• S-Adenosylmethionine is converted to homocysteine.

  • After donation of a methyl group by S-adenosylmethionine, the resulting S- adenosylhomocysteine is hydrolyzed, yielding adenosine and homocysteine.
• Regeneration of methionine

  • Methionine is regenerated by transfer of a methyl group to homocysteine from N5-methyltetrahydrofolate.

  – catalyzed by methionine synthase

  – mediated by the coenzyme methylcobalamin, which is derived from vitamin B12
• deficiency of vitamin B12 or folic acid can lead to increased homocysteine levels.
• High homocysteine levels correlate with vascular disease

  • Elevated serum levels of homocysteine or the disulfide-linked dimer homocystine are a 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.

  – are implicated in the development of type 2 diabetes.
• Formation of S-adenosylmethionine activates the methyl group of methionine, which the folate cycle restores.
• Nucleotides can be synthesized by de novo or salvage pathways

  • Nucleotides are key biomolecules.

  – Activated precursors of nucleic acids.

  – ATP is the universal currency of energy.

  – GTP serves as an energy source.

  – Nucleotide derivatives participate in biosynthetic processes.

  – CyclicAMP and cyclicGMP are essential components of signal- transduction pathways.

  – ATP acts as the donor of phosphoryl groups transferred by protein kinases.
• Nucleotides can be synthesized by de novo and salvage pathways.

  DE NOVO: activated ribose (PRPP) + amino acids + ATP + CO2 --> nucleotide

  SALVAGE PATHWAY: activated ribose (PRPP) + base --> nucleotide
• 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, the framework for the base is assembled first and then attached to ribose.

  – For purines, the base is synthesized piece by piece directly onto a ribose-based structure.

  • Salvage pathways = pathways in which preformed bases are recovered and reconnected to a ribose unit.
• The pyrimidine ring is assembled de novo from CO2, ammonia, and aspartate.

  • Pyrimidine rings are assembled from bicarbonate, aspartate, and ammonia.
  • Glutamine often serves as an ammonia donor.
• The formation of PRPP

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

  • synthesized by 5-Phosphoribosyl-1-pyrophosphate synthetase
• Glutamine provides the nitrogen via ammonia for the committed step in purine synthesis

  • Purine bases are assembled already attached to the ribose ring.

  • The committed step in purine biosynthesis forms 5-phosphoribosyl-1-amine from PRPP and glutamine.

  – catalyzed by glutamine phosphoribosyl amidotransferase
• Purine nucleotide synthesis require one- carbon units from the folate cycle.
• Two of the enzymes in the de novo purine biosynthesis pathway require 10-formyl-THF.
• The first chemotherapeutic agents were antifolates.

  • 16 children with acute lymphoblastic leukaemia (AML) with aminopterin, an amino derivative of folic acid.

  • 10 of the patients resulted in temporary remission.

  • This trial was considered the first ever successful remission of leukaemia and the foundation of modern chemotherapy.

  • Aminopterin was later later developed to methotrexate which is less toxic and is commonly used today in chemotherapy treatment.
• Methotrexate is a folic acid derivative

  • A chemotherapeutic agent in many types of cancer

  • An immunosuppressant used to used to treat inflammatory conditions, including rheumatoid arthritis.
• Methotrexate is a DHFR inhibitor

  • Methotrexate inhibits dihydrofolate reductase (DHFR)

  • This prevents generation of THF to carry one-carbon units for purine synthesis
• Many different chemical forms of antifolates target different enzymes.
• Serine and glycine are required for purine nucleotide synthesis.