Detailed Notes on Nucleotide Metabolism

Metabolism of Nucleotides

Student Learning Outcomes

State the origin of the atoms in the purine and pyrimidine rings.
Describe the de novo biosynthesis of purine and pyrimidine nucleotides.
Explain the salvage pathway for purine nucleotide biosynthesis.
Explain the conversion of ribonucleotide to deoxyribonucleotide.
Describe the catabolism of purine and pyrimidine nucleotide.

Nucleotides

Composed of a nitrogenous base, a sugar, and a phosphate group.
Nucleoside: base + sugar.
Nucleotide: base + sugar + phosphate.
Nitrogenous bases include Adenine, Guanine, Cytosine, Thymine, and Uracil. These bases form hydrogen bonds with each other.

Nucleic Acid Bases

Purines: Adenine, Guanine (double-ring structure).
Pyrimidines: Cytosine, Thymine, Uracil (single-ring structure).
These bases are planar, aromatic, and heterocyclic.
Numbering of bases is unprimed.

Sugars

Pentoses (5-C sugars).
D-Ribose and 2'-Deoxyribose.
Deoxyribose lacks a 2'-OH group.
Numbering of sugars is primed.

Nucleosides

Formed by linking a sugar with a purine or pyrimidine base through an N-glycosidic linkage.
Purines bond to the C1' carbon of the sugar at their N9 atoms.
Pyrimidines bond to the C1' carbon of the sugar at their N1 atoms.

Nucleotides

Formed by linking one or more phosphates with a nucleoside onto the 5' end of the molecule through esterification.
Esterification: combining an organic acid (RCOOH) with an alcohol (ROH) to form an ester (RCOOR) and water.

RNA vs. DNA

RNA (ribonucleic acid) is a polymer of ribonucleotides.
DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides.
Both contain Adenine, Guanine, and Cytosine.
Ribonucleotides contain Uracil.
Deoxyribonucleotides contain Thymine.
Nucleoside Triphosphates (ATP, GTP) are important energy carriers.
Important components of coenzymes like FAD, NAD+, and Coenzyme A.

Phosphate Groups

Mono-, di-, or triphosphates.
Phosphates can be bonded to either C3' or C5' atoms of the sugar.

Naming Conventions

Purine nucleosides end in "-sine" (Adenosine, Guanosine).
Pyrimidine nucleosides end in "-dine" (Thymidine, Cytidine, Uridine).
Nucleotides: Start with the nucleoside name and add "mono-", "di-", or "triphosphate" (Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate).

Nucleotide Metabolism

Purine Ribonucleotides

Formed de novo (purines are not initially synthesized as free bases).
The first purine derivative formed is Inosine Mono-phosphate (IMP) through an 11-step synthesis.
The purine base in IMP is hypoxanthine.
AMP and GMP are formed from IMP.

Purine Nucleotide Synthesis

Atoms in the purine ring come from:
- N1: Aspartate Amine.
- C2, C8: Formate.
- N3, N9: Glutamine.
- C4, C5, N7: Glycine.
- C6: Bicarbonate Ion.
ATP is involved in 6 steps.
Phosphoribosyl-alpha-pyrophosphate (PRPP) is a precursor for Purine Synthesis, Pyrimidine Synthesis, Histidine, and Tryptophan synthesis.
Role of ATP in the first step is unique – group transfer rather than coupling.
In the second step, the C1 notation changes from alpha to beta.
In step 2, PPi is hydrolyzed to 2Pi (irreversible, "committing" step).

Coupling of Reactions

Hydrolyzing a phosphate from ATP releases energy: $\Delta G°’= -30.5 \text{ kJ/mol}$ .
The energy must be coupled to an exergonic reaction.
When ATP is a reactant, part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl, or adenosinyl group.
ATP hydrolysis can drive an otherwise unfavorable reaction (synthetase; "energase").
Most active in the liver.

IMP Conversion

IMP can be converted to GMP or AMP through de novo synthesis pathways.

Regulatory Control of Purine Nucleotide Biosynthesis

GTP is involved in AMP synthesis.
ATP is involved in GMP synthesis.
PRPP is a biosynthetically "central" molecule.
ADP/GDP levels provide negative feedback on Ribose Phosphate Pyrophosphokinase.
Amidophosphoribosyl transferase is activated by PRPP levels.
APRT activity is subject to negative feedback at two sites:
- ATP, ADP, AMP bound at one site.
- GTP, GDP, and GMP bound at the other site.
Rate of AMP production increases with the concentration of GTP; rate of GMP production increases with the concentration of ATP (reciprocal control).
Above the level of IMP production:
- Independent control.
- Synergistic control.
- Feedforward activation by PRPP.
Below the level of IMP production:
- Reciprocal control.
Total amounts of purine nucleotides controlled.
Relative amounts of ATP, GTP controlled.

Purine Catabolism and Salvage

All purine degradation leads to uric acid.
Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases and intestinal phosphodiesterases in the intestine.
Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides.
Direct absorption of nucleosides.
Further degradation:
- $\text{Nucleoside} + H_2O \rightarrow \text{base} + \text{ribose (nucleosidase)}$
- $\text{Nucleoside} + P_i \rightarrow \text{base} + \text{r-1-phosphate (n. phosphorylase)}$
Most ingested nucleic acids are degraded and excreted.

Intracellular Purine Catabolism

Nucleotides are broken into nucleosides by the action of 5'-nucleotidase (hydrolysis reactions).
Purine nucleoside phosphorylase (PNP):
- $\text{Inosine} \rightarrow \text{Hypoxanthine}$
- $\text{Xanthosine} \rightarrow \text{Xanthine}$
- $\text{Guanosine} \rightarrow \text{Guanine}$
- Ribose-1-phosphate splits off and can be isomerized to ribose-5-phosphate.
Adenosine is deaminated to Inosine (ADA).
Xanthine is the point of convergence for the metabolism of the purine bases.
$\text{Xanthine} \rightarrow \text{Uric acid}$
Xanthine oxidase catalyzes 2 reactions.
The purine ribonucleotide degradation pathway is the same for purine deoxyribonucleotides.

Adenosine Degradation

AMP is converted to Adenosine or IMP.
Adenosine is converted to Inosine via Adenosine Deaminase.
IMP is converted to Inosine.

Xanthosine Degradation

The ribose sugar gets recycled (Ribose-1-Phosphate → R-5-P) and can be incorporated into PRPP.
Hypoxanthine is converted to Xanthine by Xanthine Oxidase.
Guanine is converted to Xanthine by Guanine Deaminase.
Xanthine gets converted to Uric Acid by Xanthine Oxidase.

Xanthine Oxidase

A homodimeric protein.
Contains electron transfer proteins (FAD, Mo-pterin complex, two 2Fe-2S clusters).
Transfers electrons to O2 → H2O2 (2 TIMES).
H2O2 is toxic and is disproportionated to H2O and O2 by catalase.

The Purine Nucleotide Cycle

$\text{AMP} + H<em>2O \rightarrow \text{IMP} + NH</em>4^+ \text{ (AMP Deaminase)}$
$\text{IMP} + \text{Aspartate} + \text{GTP} \rightarrow \text{AMP} + \text{Fumarate} + \text{GDP} + P_i \text{ (Adenylosuccinate Synthetase)}$
Combining the two reactions:
- $\text{Aspartate} + H<em>2O + \text{GTP} \rightarrow \text{Fumarate} + \text{GDP} + P</em>i + NH_4^+$
The overall result of combining reactions is deamination of Aspartate to Fumarate at the expense of a GTP.

Purine Salvage

Adenine phosphoribosyl transferase (APRT):
- $\text{Adenine} + \text{PRPP} \rightarrow \text{AMP} + \text{PPi}$
Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT):
- $\text{Hypoxanthine} + \text{PRPP} \rightarrow \text{IMP} + \text{PPi}$
- $\text{Guanine} + \text{PRPP} \rightarrow \text{GMP} + \text{PPi}$
These are all reversible reactions.
AMP, IMP, and GMP do not need to be resynthesized de novo!

Uric Acid Excretion

Humans excrete uric acid into urine as insoluble crystals.
Birds, terrestrial reptiles, some insects excrete insoluble crystals in paste form (conserves water).
Others further modify uric acid:
- $\text{Uric Acid} \rightarrow \text{Allantoin} \rightarrow \text{Allantoic Acid} \rightarrow \text{Urea} \rightarrow \text{Ammonia}$

Gout

Impaired excretion or overproduction of uric acid.
Uric acid crystals precipitate into joints (Gouty Arthritis), kidneys, ureters (stones).
Lead impairs uric acid excretion – lead poisoning from pewter drinking goblets.
Xanthine oxidase inhibitors inhibit the production of uric acid and treat gout.
Allopurinol treatment – a hypoxanthine analog that binds to Xanthine Oxidase to decrease uric acid production.

Allopurinol

A xanthine oxidase inhibitor.
A substrate analog is converted to an inhibitor, a “suicide-inhibitor."

Lesch-Nyhan Syndrome

A defect in the production or activity of HGPRT.
Causes increased levels of Hypoxanthine and Guanine, which leads to increased degradation to uric acid.
PRPP accumulates, stimulating the production of purine nucleotides (and thereby increases their degradation).
Causes gout-like symptoms and neurological symptoms (spasticity, aggressiveness, self-mutilation).
The first neuropsychiatric abnormality attributed to a single enzyme.

Purine Autism

25% of autistic patients may overproduce purines.
To diagnose, urine must be tested over 24 hours (biochemical findings disappear in adolescence).
Pink urine due to uric acid crystals may be seen in diapers.

Pyrimidine Ribonucleotide Synthesis

Uridine Monophosphate (UMP) is synthesized first.
CTP is synthesized from UMP.
Pyrimidine ring synthesis is completed first, then attached to ribose-5-phosphate.
- N1, C4, C5, C6: Aspartate.
- C2 : HCO3-.
- N3 : Glutamine amide Nitrogen.

Pyrimidine Synthesis

2 ATP+HCO3 +Glutamine+H2O→Carbamoyl Phosphate
$\text{Carbamoyl Phosphate} + \text{Aspartate} \rightarrow \text{Carbamoyl Aspartate}$
$\text{Carbamoyl Aspartate} \rightarrow \text{Dihydroorotate}$
$\text{Dihydroorotate} \rightarrow \text{Orotate}$
$\text{Orotate} + \text{PRPP} \rightarrow \text{Orotidine-5'-monophosphate (OMP)}$
$\text{OMP} \rightarrow \text{Uridine Monophosphate (UMP)}$

UMP Synthesis Overview

2 ATPs are needed (both used in the first step).
One transfers phosphate, the other is hydrolyzed to ADP and Pi.
2 condensation reactions form carbamoyl aspartate and dihydroorotate.
Dihydroorotate dehydrogenase is an intramitochondrial enzyme; oxidizing power comes from quinone reduction.
The attachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-P.
PPi splits off PRPP – irreversible.
Channeling: enzymes 1, 2, and 3 are on the same chain; 5 and 6 are on the same chain.

OMP Decarboxylase

The final reaction of the pyrimidine pathway.
Another mechanism for decarboxylation.
A high-energy carbanion intermediate is not needed.
No cofactors are needed!
Some of the binding energy between OMP and the active site is used to stabilize the transition state.
"Preferential transition state binding."

UMP → UTP and CTP

Nucleoside monophosphate kinase catalyzes the transfer of Pi to UMP to form UDP; nucleoside diphosphate kinase catalyzes the transfer of Pi from ATP to UDP to form UTP.
CTP is formed from UTP via CTP Synthetase, driven by ATP hydrolysis.
Glutamine provides amide nitrogen for C4 in animals.

Regulatory Control of Pyrimidine Synthesis

Differs between bacteria and animals.
Bacteria – regulation at ATCase rxn.
Animals – regulation at carbamoyl phosphate synthetase II.
UDP and UTP inhibit the enzyme; ATP and PRPP activate it.
UMP and CMP competitively inhibit OMP Decarboxylase.
Purine synthesis is inhibited by ADP and GDP at ribose phosphate pyrophosphokinase step, controlling the level of PRPP.

Orotic Aciduria

Caused by a defect in the protein chain with enzyme activities of the last two steps of pyrimidine synthesis.
Increased excretion of orotic acid in urine.
Symptoms: retarded growth, severe anemia.
Only known inherited defect in this pathway (all others would be lethal to the fetus).
Treat with uridine/cytidine.

Degradation of Pyrimidines

CMP and UMP are degraded to bases similarly to purines.
Dephosphorylation.
Deamination.
Glycosidic bond cleavage.
Uracil is reduced in the liver, forming beta-alanine.
Converted to malonyl-CoA → fatty acid synthesis for energy metabolism.

Deoxyribonucleotide Formation

Purine/Pyrimidine degradation is the same for ribonucleotides and deoxyribonucleotides.
Biosynthetic pathways are only for ribonucleotide production.
Deoxyribonucleotides are synthesized from corresponding ribonucleotides.

Formation of Deoxyribonucleotides

Reduction of the 2' carbon is done via a free radical mechanism catalyzed by "Ribonucleotide Reductases."
E. coli RNR reduces ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs).
Two subunits: R1 and R2 (Heterotetramer: (R1)2 and (R2)2 in vitro).

R1 Subunit

Three allosteric sites (Specificity Site, Hexamerization site, Activity Site).
Five redox-active –SH groups from cysteines.

R2 Subunit

Tyr 122 radical - close to the Fe(III) complex.
Binuclear Fe(III) complex (Fe prosthetic group).
Fe's are bridged by O-2 and carboxyl group of Glu 115.

Mechanism of Ribonucleotide Reductase Reaction

Free Radical.
Involvement of multiple –SH groups.
RR is left with a disulfide group that must be reduced to return to the original enzyme.

Anti-Folate Drugs

Cancer cells consume dTMP quickly for DNA replication.
Interfere with thymidylate synthase rxn to decrease dTMP production.
(fluorodeoxyuridylate – irreversible inhibitor) affects rapidly growing normal cells (hair follicles, bone marrow, the immune system, intestinal mucosa).
The Dihydrofolate reductase step can be stopped competitively (DHF analogs).
Anti-Folates: Aminopterin, methotrexate, trimethoprim.

Adenosine Deaminase Deficiency

The second most common form of SCID after X-SCID is caused by a defective enzyme, adenosine deaminase (ADA), necessary for the breakdown of purines.
Lack of ADA causes accumulation of dATP (inhibits Ribonucleotide Reductase).
Without functional ribonucleotide reductase, lymphocyte proliferation is inhibited, and the immune system is compromised.

Adenosine Deaminase Deficiency (ADA)

In purine degradation, Adenosine → Inosine (Enzyme is ADA).
ADA deficiency results in SCID - "Severe Combined Immunodeficiency." Selectively kills lymphocytes (both B and T cells).
All known ADA mutants structurally perturb the active site.

Adenosine Deaminase & Gene Therapy

One of the first diseases to be treated with gene therapy.
The ADA gene is inserted into lymphocytes, then lymphocytes are returned to the patient.
PEG-ADA treatments (activity lasts 1-2 weeks).

Here are some of the important points covered in the provided text:

Purine and Pyrimidine Biosynthesis: Understanding the de novo synthesis pathways for purine and pyrimidine nucleotides, including the origin of atoms in the rings.
Regulation of Nucleotide Biosynthesis: Grasping the regulatory mechanisms that control purine and pyrimidine synthesis, including feedback inhibition and activation.
Purine and Pyrimidine Catabolism: Knowing how purines and pyrimidines are broken down.
Salvage Pathways: Understanding how purine bases are salvaged to form nucleotides, and the enzymes involved (e.g., APRT, HGPRT).
Conversion of Ribonucleotides to Deoxyribonucleotides: Learning how ribonucleotides are converted to deoxyribonucleotides via ribonucleotide reductase.
Clinical Relevance: Understanding the clinical implications of nucleotide metabolism disorders like gout, Lesch-Nyhan syndrome, ADA deficiency, and orotic aciduria, including treatments and genetic aspects.