Vet Biochem: Unit 1-3 (ch 1-19)

• Nucleotides

  • functions

  • building block of DNA and RNA
  • used in energy transduction reactions
  • act as activated carriers of carbohydrates, amino acids, lipids, sulfates, and methyl groups
  • structural components of coenzymes (coenzyme A, NAD, FAD)
  • regulators of key intracellular enzymes (cAMP, cGMP
• Nucleotides

  • origin / synthesis

  • from small organic molecules available in cells
  • through salvage pathways that recycle nitrogen bases
• Nucleotide Structure

  • nucleotides are composed of:

  • nitrogen base (purine / pyrimidine)
  • cyclic pentose
  • phosphate groups
  • nucleoside: nitrogen base + pentose
  • nucleotide: nucleoside + phosphate
• nucleotide groups

  • purines: adenine, guanine
  • pyrimidines: cytosine, uracil (RNA only), thymine (DNA only)
• DNA vs. RNA

  • RNA (ribonucleic acid) has ribose group attached to OH at 2’ carbon atom
  • DNA (deoxyribonucleic acid) has ribose group attached to H at 2’ carbon atom
• purine and pyrimidine nucleosides are derived from common precursors

  • glutamine (Gln), carbon dioxide (CO2 ), aspartate (Asp), 5-phosphoribosyl-1- pyrophosphate (PRPP), and, for thymine nucleotides, tetrahydrofolate derivatives
• Pathway summary:

  • phase 1: CAP formation

  • carbamoyl phosphate is formed from Gln, ATP, and CO2 , catalyzed by cytoplasmic CPS-2

  
• Pathway Summary

  • phase 2: carbamoyl aspartic acid formation

  • CAA is formed by condensation of CAP with Asp through Aspartate Transcarbamoylase

  
• Pathway Summary

  • phase 3: ring closure, DHOA formation

  • closure is through loss of H2O, facilitated by dihydroorotase

  
• Pathway Summary

  • phase 4: OMP formation

  • ribose phosphate is transferred from PRPP to DHOA, forming OMP

  
• • phase 5: OMP decarboxylation

  forms UMP, the first pyrimidine

  
• phase 5: OMP decarboxylation

  • complete when UTP is aminated to become CTP by Gln and ATP

  
• • all organisms except protozoa synthesize purines
  • synthetic pathway has much in common with pyrimidine NTPs 
  • ribose sugar and a-phosphate are generated from 5'-phosphoribosyl-1-pyrophosphate (PRPP)
  • b- and g-phosphates are generated from ATP breakdown and from the TCA cycle
• purine structure

  
• PURINE BIOSYNTHESIS:

  Phase 1: PRPP Biosynthesis

  • PRPP is synthesized from ribose-5-phosphate and peATP by PRPP synthetase 
  • the availability of PRPP in the cell is tightly controlled via allosteric "negative feedback" regulation of PRS activity by various purine nucleotides produced in later stages of the pathway, namely IMP, AMP, GMP, and ADP
• PURINE BIOSYNTHESIS

  Phase 2: IMP formation

  • step 1: amino group is added to PRPP (PRA)
  • step 2: PRA condensation with Gly (glycinamide ribosyl-5-phosphate) (ring C 4-5, ring N-7)
  • step 3: methenyl-tetrahydrofolate donates formyl group to form formyl glycinamide ribosyl-5-phosphate
• PURINE BIOSYNTHESIS

  Phase 2: IMP formation

  • step 5: dehydration closes ring to form aminoimidazole ribosyl-5-phosphate
  • step 6: ATP and biotin-independent carboxylation yields aminoimidazole carboxylate ribosyl-5-phosphate (ring C-6)
  • step 7: condensation of Asp with aminoimidazole carboxylate ribosyl-5- phosphate followed by dehydration and release of fumarate then forms aminoimidazole carboxamide ribosyl-5- phosphate
• PURINE BIOSYNTHESIS

  Phase 2: IMP formation

  • step 8: ring carbon 2 is added, forming formimidoimidazole carboxamide ribosyl-5- phosphate 
  • step 9: dehydration and ring closure, forming parent purine nucleotide, inosine monophosphate (IMP)  
• PURINE BIOSYNTHESIS

  Phase 3: Formation of AMP. GMP, and 5’-triphosphates

  • “A” branch yields AMP, “G” branch yields GMP
• Phase 3: A Branch

  • conversion of IMP to AMP by replacement of 6-ketogroup with amino group
  • step 1: keto group of IMP reacts with the amino group of Asp to form adenylosuccinate (AMPS), using adenylosuccinate synthetase 
  • step 2: adenylosuccinase catalyzes the release of fumarate from AMPS, thus yielding AMP 
• Phase 3: G Branch

  • conversion of IMP to GMP by replacement of 6-ketogroup with amino group
  • step 1: NAD+-dependent oxidation of the 2 carbon by IMP dehydrogenase, which generates the 2,6-diketo derivative, xanthosine monophosphate (XMP), and NADH
  • step 2: amination of the 2-keto group of XMP to form GMP, exploiting the amido group of Gln and pyrophosphorolysis of peATP as a source of energy
• folic acid (folate, pterylglutamate) structure

  • water-soluble vitamin, consisting of PABA with amino end attached to pteridine, and carboxyl end attached to a-amino group of glutamic acid 
• tetrahydrofolate (THFA, H4 folate)

  • active cofactor form of FA
  • formed by reduction of pteridine ring in positions 5, 6, 7, 8 by enzyme folic acid reductase
  • essential in metabolism because it donates carbon during purine, dTMP and methionine synthesis 
• folate is an essential substance in the body

  • folate synthesis is inherent with bacteria through synthesis using pteridine, PABA and Glu 
  • animals cannot synthesize folate, and therefore is needed to be incorporated in the diet
• folate metabolism in bacteria are exploited in pharmacology

  • ability of bacteria to assimilate FA is the basis for making bacteria-selective sulfonamide antibiotics 
  • sulfonamides are PABA analogs, which competes with PABA preventing folate synthesis
• FA absorption in GIT requires removal of extra glutamates

  • reduction requires NADPH and FAR, and proceeds in two steps, to dihydrofolate and then to THFA
  • THFA picks up the one-carbon methylene group from serine (Ser) to form N5,N10- methylene-H4 folate, used in thymine synthesis or to be used as cofactor in methionine methylation
• Thymine is the methylated form of uracil

  • important as uracil is not found in DNA of prokaryotes and animals 
  • if not synthesized, DNA synthesis cannot proceed and dTTP supply is insufficient
  • to control dTTP supply, animals have enzymes dUTPase to dephosphorylate dUTP to dUMP, and thymidylate synthase (TS) to methylate dUMP to dTMP
• nucleotides have varied fates

  • it can be incorporated to form nucleic acids 
  • it can be a carrier for sugar transport 
  • may be cyclized to form 3’-5’NMP 
  • may act as a phosphate donor 
  • the base, carbohydrate, and phosphate moities are constantly “turning over” in response to salvage and degradation
• DNA and RNA degradation occurs in two sites; intracellular or intraluminal

  • step 1: DNA/RNA degradation using endonucleases DNAse and RNAse
  • step 2: degradation by exonucleases to form 5’- and 3’-ribo and deoxyribo-NMPs  
• Thymine is the methylated form of uracil

  • important as uracil is not found in DNA of prokaryotes and animals 
  • if not synthesized, DNA synthesis cannot proceed and dTTP supply is insufficient
  • to control dTTP supply, animals have enzymes dUTPase to dephosphorylate dUTP to dUMP, and thymidylate synthase (TS) to methylate dUMP to dTMP
• NMPs from nucleic acid have two fates;

  • fate 1: ATP-dependent phosphorylation by NMP and NDP kinase to reform useful NTPs 
  • fate 2: dephosphorylation by nucleotidases and non-specific phosphatase forming ribo- and deoxyribonucleosides  
• Salvage of Purine and Pyrimidine Bases

  • base salvage is essential since they are expensive to synthesize de novo
  • salvage pathways are important in rapidly dividing cells (bone marrow, intestinal cells)
• Degradation of Pyrimidine Bases

  • products of pyrimidine degradation are water-soluble, and is reused rather than excreted
  • reuse of pyrimidine degradation products are essential in cases of starvation
• Degradation of Purine Bases

  • degradation is more complex than in pyrimidines 
  • HX, guanine, and adenine are degraded to become xanthine
• ATP is a nucleotide
• • nucleoside: nitrogen base + pentose
• • nucleotide: nucleoside + phosphate 
• • purines: adenine, guanine 
• pyrimidines: cytosine, uracil (RNA only), thymine (DNA only) 

  • Only have 1 ring/ring
  • 6 atoms, 2 N and 4 C
• • RNA (ribonucleic acid) has ribose group attached to OH at 2’ carbon atom