Nucleotide metabolism Part 1 (purine biosynthesis)

Chapter 26
The Synthesis and Degradation of Nucleotides
Biochemistry by Reginald Garrett and Charles Grisham

Introduction to Nucleic Acids Definitions . Definitions By definition, nucleic acids are biomolecules that store genetic information in cells or that transfer this information from old cells to new cells. There are two groups of nucleic acids: DeoxyRiboNucleic Acid (DNA) and RiboNucleic Acid (RNA).

Biological significance of nucleotide metabolism

Nucleotides make up nucleic acids (DNA and RNA) Nucleotide triphosphates are the energy carriers in cells (primarily ATP) Many metabolic pathways are regulated by the level of the individual nucleotides
Example: cAMP regulation of glucose release

Adenine nucleotides are components of many of the coenzymes

Examples: NAD+, NADP+, FAD, FMN, coenzyme A

Dietary nucleotides
do not contribute energy as do carbs, proteins and fats are not incorporated into RNA or DNA unless given I.V. normally metabolized to individual components (bases, sugar and phosphate) purines are converted to uric acid which is then excreted

Medical significance of nucleotide metabolism

Anticancer agents:
Rapidly dividing cells biosynthesize lots of purines and pyrimidines, but other cells reuse them. Cancer cells are rapidly dividing, so inhibitor of nucleotide metabolism kill them

Antiviral agents
Zidovudine (Retrovir) Lamivudine (Epivir) Valacyclovir (Valtrex)

Structures of nucleotide building blocks and nucleotides

6 5 1 N 2 N 3 PURINE 4 N 8 N 9 H 7 3N 2 N 1 4 5 6

PYRIMIDINE

Structures of nucleotide building blocks and nucleotides

NH2 N N ADENINE N N H2N N O N HN O N H THYMINE O CH3 HN O N H URACIL
N O N H CYTOSINE

NH2

N H

N H

GUANINE

guanine: comes from guano; thymine thymus gland

OH OH P O H OH OH O CH 2 H Base O H H OH

OH P O H OH H O CH 2 H Base O H H

RIBONUCLEOTIDE

DEOXYRIBONUCLEOTIDE

Ribonucleotide phosphate = ribonucleoside

Biosynthesis of the purine nucleotide system

Synthesis of Inosine Monophosphate

Basic pathway for biosynthesis of purine ribonucleotides Starts from ribose-5-phosphate which is derived from the pentose phosphate pathway Requires 11 steps overall occurs primarily in the liver

Nucleotide Metabolism
PURINE RIBONUCLEOTIDES: formed de novo
i.e., purines are not initially synthesized as free bases First purine derivative formed is Inosine Monophosphate (IMP)
The purine base is hypoxanthine AMP and GMP are formed from IMP

Purine Nucleotides
Get broken down into Uric Acid (a purine) Buchanan (mid 1900s) showed where purine ring components came from:

N1: Aspartate Amine C2, C8: Formate N3, N9: Glutamine C4, C5, N7: Glycine C6: Bicarbonate Ion

Purine Nucleotide Synthesis

COO

O C

OOC
2-

O3P O CH2 H H

O H

H OH

C4 C H2N
5

N CH N

Aspartate + ATP

ADP + Pi

HC CH2 COO

N H

C4 C H2N
5

N CH N

OH OH -D-Ribose-5-Phosphate (R5P)
ATP
Ribose Phosphate Pyrophosphokinase

SAICAR Synthetase

Ribose-5-Phosphate Carboxyamidoimidazole Ribotide (CAIR)

AIR Car boxylase ADP + Pi

Ribose-5-Phosphate 5-Aminoimidazole-4-(N-succinylocarboxamide) ribotide (SAICAR)

Fumarate
Adenylosuccinate Lyase

AMP

ATP +HCO3

O C

2-

O3P O CH2 H H OH

O H OH

H O

O P O O

O P O O

HC 4 C H2N
5

N CH N
H2N

C C H2N

N
4 5

CH N

Ribose-5-Phosphate
5-Phosphoribosyl--pyrophosphate (PRPP)

Ribose-5-Phosphate 5-Aminoimidazole-4-carboxamide ribotide (AICAR)

N10-FormylTHF

5-Aminoimidazole Ribotide (AIR)

ADP + Pi AIR Synthetase

Glutamine + H2O Glutamate + PPi

Amidophosphoribosyl Transferase

ATP

H N H2C CH O NH
O C H

O THF C H2N C C NH

AICAR Transformylase

2-

O3P O

CH2 H H OH

O H

NH2

N
4 5

C HN

CH N

H OH

-5-Phosphoribosylamine (PRA)
Glycine + ATP
GAR Synthetase

Ribose-5-Phosphate Formylglycinamidine ribotide (FGAM)

FGAM Synthetase ADP + Glutamate + Pi ATP + Glutamine + H2O

Ribose-5-Phosphate 5-Formaminoimidazole-4-carboxamide ribotide (FAICAR)

H2O
IMP Cyclohydrolase

ADP + Pi

H 2C O
2-

NH2

H N H2C C

O C N
4

CH O NH
2-

HN HC N
O3P O CH2 H H OH O

C NH H H OH
GAR Transformylase

CH N
H H OH

C5

O3P O

CH2 H H OH

N10-Formyl-THF

THF

Ribose-5-Phosphate Formylglycinamide ribotide (FGAR)

Glycinamide Ribotide (GAR)

Inosine Monophosphate (IMP)

The big picture

Steps 1 thru 3
Step 1:Activation of ribose-5-phosphate
enzyme: ribose phosphate pyrophosphokinase product: 5-phosphoribosyl--pyrophosphate (PRPP) PRPP is also a precursor in the biosynthesis of pyrimidine nucleotides and the amino acids histidine and tryptophan

Step 1: purine synthesis

Steps 1 thru 3
Step 2: acquisition of purine atom 9
enzyme: amidophosphoribosyl transferase displacement of pyrophosphate group by glutamine amide nitrogen (inversion of configuration to product: -5-phosphoribosylamine
Steps 1 and 2 are tightly regulated by feedback inhibition

Step 2: purine synthesis:

commited step

Steps 1 thru 3
Step 3: acquisition of purine atoms C4, C5, and N7
enzyme: glycinamide synthetase -phosphoribosylamine reacts with ATP and glycine product: glycinamide ribotide (GAR)

Step 3 : purine synthesis

Steps 4 thru 6
Step 4: acquisition of purine atom C8
formylation of free -amino group of GAR enzyme: GAR transformylase co-factor of enzyme is N10-formyl THF

Step 5: acquisition of purine atom N3

The amide amino group of a second glutamine is transferred to form formylglycinamidine ribotide (FGAM)

Step 6: closing of the imidazole ring or formation of 5aminoimidazole ribotide

Step 6: purine synthesis

Step 7
Step 7: acquisition of C6
C6 is introduced as HCO3 enzyme: AIR carboxylase (aminoimidazole ribotide carboxylase) product: CAIR (carboxyaminoimidazole ribotide) enzyme composed of 2 proteins: PurE and PurK (synergistic proteins)

Step 7: purine synthesis

Steps 8 thru 11
Step 8: acquisition of N1
N1 is acquired from aspartate in an amide condensation reaction enzyme: SAICAR synthetase product: 5-aminoimidazole-4-(Nsuccinylocarboxamide)ribotide (SAICAR) reaction is driven by hydrolysis of ATP

Step 8: purine synthesis

Steps 8 thru 11
Step 9: elimination of fumarate
Enzyme: adenylosuccinate lyase Product: 5-aminoimidazole-4-carboxamide ribotide (AICAR)

Step 10: acquisition of C2

Another formylation reaction catalyzed by AICAR transformylase Product: 5-formaminoimidazole-4carboxamide ribotide (FAICAR)

Step 9: purine synthesis

Step 10: purine synthesis

Step 11
cyclization or ring closure to form IMP water is eliminated in contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells

Step 11: purine synthesis

O HN N IMP A.S. synthetase COO-OOC NH N N N O N Ribose-P Adenylosuccinate xanthine monophosphate XMP glutamine + ATP A.S. lyase fumarate NH 2 N N N Glutamate + AMP + PPi O HN H2 N N N N H HN N

N Ribose-P IMP dehydrogenase

Synthesis of adenine and guanine nucleotides

Aspartate + GTP GDP

NAD +

NADH O N

N Ribose-P

N Ribose-P

N Ribose-P

AMP

GMP

Purine nucleoside diphosphates and triphosphates: - to be incorporated into DNA and RNA, nucleoside monophosphates (NMPs) must be converted into nucleoside triphosphates (NTPs) - nucleoside monophosphate kinases (adenylate & guanylate kinases)
AMP + ATP 2 ADP accomplished by separate enzymes GMP + ATP GDP + ADP

- nucleoside diphosphate kinase

GDP + ATP GTP + ADP

same enzyme acts on all nucleotide di & triphosphates nucleoside diphosphate k inase is an enzyme which plays a k ey role in the activation of antiviral nucleosides such as Retrovir/A

Regulation of purine nucleotide biosynthesis

activation amidophosphoribosyl transferase PRPP 5-P-ribosylamine ribose-P-pyrophosphok inase Ribose-5-P

AMP + GMP

ADP + GDP

The purine salvage pathway

Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides Two phosphoribosyl transferases are involved:
APRT (adenine phosphoribosyl transferase) for adenine HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine

Salvage of purines
OH OH P O H OH OH O CH2 H H O H O O P OO O P OOPPi adenine

NH 2 N N N

Adenine phosphoribosyltransferase (APRT)

OH OH P O H OH OH O CH2 H O H

Salvage of purines
Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in neural tissue) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Hypoxanthine + PRPP IMP + Ppi Guanine + PRPP GMP + Ppi Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cells

Salvage of purine bases

Inborn Errors Of Purine and Pyrimidine matabolsim

Severe combined immunodeficiency syndrome (SCID)

Xanthine Oxidase and Gout Xanthine Oxidase in liver, intestines mucosa, and milk can oxidize hypoxanthine to xanthine and xanthine to uric acid Humans and other primates excrete uric acid in the urine, but most N goes out as urea Birds, reptiles and insects excrete uric acid and for them it is the major nitrogen excretory compound Gout occurs from accumulation of uric acid crystals in the extremities Allopurinol, which inhibits XO, is a treatment

Regulation of pyrimidine biosynthesis

In bacteria
allosterically inhibited at ATCase by CTP (or UTP) allosterically activated at ATCase by ATP (compete with CTP)

In animals
UDP and UTP are feedback inhibitors of CPS II PRPP and ATP are allosteric activators