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

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

Overview of digestion and utilization of nucleic acids

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.

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

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)

FH4 (or THF) N10—CHO—FH4

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

Source For Ribose-5-Phosphate

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

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)

Step 1:Activation of ribose-5-phosphate OH 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

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

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-)

Step 4: acquisition of purine atom C8 GAR transformylase

Step 5: acquisition of purine atom N3

Step 6: closing of the imidazole ring

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

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

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

adenylosuccinate lyase Step 9: elimination of fumarate adenylosuccinate lyase

Step 10: acquisition of C2 AICAR transformylase

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

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.

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

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.

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