1. De Nova synthesis of Purine Nucleotides
Dr. Shumaila Asim
Lecture # 3

2. Biosynthesis of Purines
Very little or no dietary purines and pyrimidines are converted into nucleotides or incorporated into nucleic acids
Dietary nucleic acids / nucleotides are converted into purine/ pyrimidine bases which are then completely metabolized for excretion
Human tissues synthesize purines and pyrimidines from amphibolic intermediates according to the physiologic need of cell / tissue

3. Overview of digestion and utilization of nucleic acids

4. There are two pathways leading to nucleotides
De novo synthesis: The synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and one-carbon units.
Salvage pathways: The synthesis of nucleotide by recycle the free bases or nucleosides released from nucleic acid breakdown.

6. De novo Synthesis of Purine Nucleotides
Precursors
Amino acids (Glycine, Aspartate, Glutamine)
CO2 (from HCO3-)
Formyl group (from formyl-tetra-hydro-folate)
Ribose-5-phosphate (HMP shunt & nucleosides)
Enzymes (cytosol)
Coenzymes and Cofactors
Formyl-and methenyl-tetra-hydro-folate, Mg++
ATPs
For Energy & PRPP formation

7. Sources of Atoms of Purine Nucleus
The image shows the source of different atoms in a purine skeleton identified by radio labeling studies
N1 is derived from amino group of Aspartate
C2 & C8 is derived from Formate
N3 & N9 is derived from amide group of Glutamine
C4, C5 & N7 is derived from Glycine
C6 is derived from HCO3- (bicarbonate)

8. FH4 (or THF)
N10—CHO—FH4

9. De novo purine synthesis
The purine ring is synthesized by a series of reactions that add the carbon and nitrogen atoms to a pre-formed ribose-5-phosphate
Starts from ribose-5-phosphate(R-5-P) The ribose-5-phosphate is synthesized as part of the Hexose MonoPhosphate pathway.
Requires 11 steps overall
occurs primarily in the liver

10. Source For Ribose-5-Phosphate

11. Formation of PRPP - + Regulation by feed back inhibitionPi
ADP & 2,3-BPG -
Regulation by feed back inhibition
PRPP-synthase and PRPP-glutamyl-amido-tranferase are
inhibited by IMP, GMP & AMP

12. Step-1: Ribose-5-phosphate activation and formation of PRPP): α- D-Ribose-phosphate (R5P) is activated with ATP to form 5- phosphoribosyl-α-pyrophosphate (PRPP) with the help of enzyme Ribose phosphate pyrophosphokinase.
Step-2: Acquisition of N9 atom of purine: Amide nitrogen of glutamine displaces the pyrophosphate group of PRPP to form β-5- phosphoribosylamine (PRA) with the help of enzyme amidophosphoribozyl transferase. (This reaction contribute N9 atom of purine form glutamine)
Step-3: Acquisition of C4, C5 & N7 atoms of purine: Carboxylic group of glycine is combined with the amino group of β-5- phosphoribosylamine (PRA) to form glycinamide ribotide (GAR) with the help of enzyme – GAR synthetase (C4, C5, & N7 of purine are contributed by glycine)

13. Step 1:Activation of ribose-5-phosphateOH 1
ATP
AMP
Step 1:Activation of ribose-5-phosphate
Committed step
ribose phosphate pyrophosphokinase 2
Step 2: acquisition of purine atom N9
Gln:PRPP amidotransferase
Steps 1 and 2 are tightly regulated by feedback inhibition
5-PRA

14. Step 3: acquisition of purine atoms C4, C5, and N7
glycinamide synthetase

15. Step-4: Acquisition of C8 atom of purine: Amino group of glycinamide ribotide (GAR) is formylated with N10- formyltetrahydrofolate and forms formylglycinamide ribotide (FGAR) with the presence of enzyme GAR transformylase. (C8 of purine is contributed by formate)
Step-5: Acquisition of N3 atom of purine: Amide nitrogen of second glutamine is added to FGAR in an ATP-dependent reaction to form formylglycinamidine ribotide (FGAM) with the help of enzyme FGAM synthetase. (N3 of purine is contributed by glutamine)
Step-6: Purine imidazole ring formation: An ATP dependent ring closing (imidazole ring formation) reaction in the presence of AIR synthetase enzyme to produce 5-aminoimidazole ribotide (AIR).
Step-7: Acquisition of C6 atom of purine: An ATP dependent carboxylation reaction of 5-aminoimidazole ribotide (AIR) with HCO3- (bicarbonate) to produce carboxyaminoimidazole ribotide (CAIR) in the presence of enzyme AIR carboxylase. (C6 of purine is contributed by HCO3-)

16. Step 4: acquisition of purine atom C8
GAR transformylase

17. Step 5: acquisition of purine atom N3

18. Step 6: closing of the imidazole ring

19. Carboxyaminoimidazole ribonucleotide (CAIR) 7
Step 7: acquisition of C6
Carboxyaminoimidazole
ribonucleotide (CAIR) 7
AIR carboxylase

20. Step-8: Acquisition of N1 atom of purine: Aspartate is added and it forms an amide bond with C6 to form 5-aminoimidazole-4-(N- succinylocarboxamide) ribotide (SACAIR) in an ATP dependent reaction with the help of enzyme SAICAR synthetase (N1 of purine is contributed by aspartate)
Step-9: Elimination of fumarate: Fumarate group is cleaved off from SACAIR to produce 5-aminoimidazole-4-carboxamide ribotide (AICAR) with the help of enzyme- adenylosuccinate lyase.
Step-10: Acquisition of C2 atom of purine: Amino group of AICAR react with N10-formyltetrahydrofolate (formylation) to form 5- formaminoimidazole-4-carboxamide ribotide (FAICAR) with presence of enzyme AICAR transformylase. (C2 of purine ring is contributed by this N10-formyltetrahydrofolate)
Step-11: Cyclization to form IMP: In the last reaction, the larger ring of FAICAR is enzymatically closed to forms Inosine Monophosphate (IMP) with the release of a water molecule catalyzed by the enzyme IMP cyclohydrolase

21. Carboxyaminoimidazole ribonucleotide (CAIR)
Step 8: acquisition of N1
Carboxyaminoimidazole
ribonucleotide (CAIR)
SAICAR synthetase

22. adenylosuccinate lyase
Step 9: elimination of fumarate
adenylosuccinate lyase

23. Step 10: acquisition of C2
AICAR transformylase

24. Step 11: ring closure to form IMP
Once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells).

26. Conversion of IMP to AMP
IMP is converted to AMP in two enzymatic steps
Step-1: Donation of amino group by aspartate: Amino group of aspartate is enzymatically linked to the IMP (C6 of purine) coupled with GTP hydrolysis to form adenylosuccinate with the help of enzyme- adenylosuccinate synthetase.
Step-2: Eliminates fumarate group to form AMP: Adenylosuccinate is enzymatically converted to AMP by the removal of fumarate group with the help of enzyme adenylosuccinate lyase.

27. Conversion of IMP to AMP and GMP
Note: GTP is used for AMP synthesis.
IMP is the precursor for both AMP and GMP.

28. Conversion of IMP to GMP
IMP is converted to GMP in two enzymatic steps
Step-1:Dehydrogenation of IMP: IMP is enzymatically dehydrogenated to form Xanthosine Monophosphate (XMP) with the enzyme IMP dehydrogenase. The H+ ions released are accepted by NAD+.
Step-2: Amidation of XMP: In the second step, XMP is amidated with the amide group from glutamine with the presence of H2O and hydrolysis of ATP yields GMP (Guanosine monophosphate); catalyzed by the enzyme GMP synthetase.

29. ADP, ATP, GDP and GTP biosynthesis
kinase
kinase
AMP
ADP
ATP
ATP
ADP
ATP
ADP
kinase
kinase
GMP
GDP
GTP
ATP
ADP
ATP
ADP