Nucleotide Metabolism Flashcards
LO #1 nucleotide metabolism
- To understand the many roles of nucleotides and their derivatives in human metabolism, you should be able to (p. 274-277; Figs. 8.3, 16.1, 16.2 and 16.3; Table 16.1):
a. Summarize the structure and function of nucleosides, nucleotides and deoxynucleotides
b. Use the differences in nomenclature to determine the constituents of the nucleic acids
i. Differentiate between purines and pyrimidines and their derivatives
c. Understand from the correlation box:
i. Specific roles of nucleosides and nucleotides (green, p. 276)
Describe the differences between Nucleotides vs. Nucleosides
•Nucleotides are phosphate esters of nucleosides
–Nucleosides = nitrogenous base + sugar
–Nucleotides = nitrogenous base + sugar + phosphate
What are dNTPs?
dNTPs (for PCR) = a mix of dATP, dCTP, dGTP, dTTP
- dATP is not equivalent to ATP (for energy)
- AMP, ADP, ATP
Polymerase chain reaction (PCR) is a method widely used in molecular biology to make many copies of a specific DNA segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
Nomenclature summary table
What are the specific roles of nucleosides and nucleotides?
Specific roles of nucleosides and nucleotides (pg. 276): nucleotides such as ATP and GTP are important cosubstrates in an array of enzymatic reactions. Nucleotides are also components of several cofactors, including CoA, FAD, FMN, UDP-Glc and NADPH and NADH.
- Nucleotides such as cAMP and cGMP play regulatory roles and serve as stabilizing regulatory elements, such as m7GTP cap at the 5’ end of eukaryotic mRNA.
- Nucleosides also appear in important biomolecules, such as adenosine in vitamin B12
- Coenzyme A
- Flavin adenine dinucleotide
- Flavin mononucleotide
- Uridine diphosphate glucose
- Nicotinamide adenine dinucleotides with and without the phosphate group
- 7-methylguanosine 5’-triphosphate
LO #2 nucleotide metabolism
- To understand the role of the de novo synthesis pathways of purines and pyrimidines in cancer and other disease states, you should be able to (p. 279-285; Figs. 16.6, 16.7 and 16.8; Table 16.2):
a. Classify the general constituents of the ring structures and the pathways that supply them.
b. Identify the stage of required use of PRPP, carbamoyl phosphate and folate derivatives in each pathway.
c. Classify important intermediates, and predict the pathways they are utilized
i. Orotate and UMP in pyrimidine nucleotide synthesis vs IMP and XMP in purine nucleotide synthesis
d. Identify the key enzymes at the regulatory checkpoints and their products
e. Understand from the correlation boxes:
i. Pentose phosphate pathway (blue, p. 276)
ii. Methotrexate (blue, p. 275)
iii. “Sulfa” drugs (blue, p. 275)
iv. Depriving cells of GMP and dGTP by antiviral agent acylovir (blue, p. 285)
Where does De Novo Synthesis of purine nucleotides occur?
De novo synthesis
Site: liver, cytosol
Biological product is produced from intermediates in the degradative pathway of its own or a similar substance.
Formation of purine base on ribose 5-phosphate from the pentose phosphate pathway
Where does the salvage pathway of purine and pyrimadine nucleotides occur?
Salvage Pathway
Site: organelles
Addition of ribose 5-phosphate to the preformed purine base
Nucleotide salvage pathways are used to recover bases/nucleosides that are formed during degradation of RNA and DNA.
Where does De Novo Synthesis of pyrimidine nucleotides occur?
De novo synthesis
Site: liver, cytosol, mitochondria
Describe purine synthesis.
- Phase I: activation of ribose 5-phosphate
- Phase II: conversion of PRPP into phosphoribosylamine*
- Phase III: construction of inosine monophosphate branch point purine ring
- Phase IV: conversion of IMP into adenosine and guanosine (deoxy) nucleotides
Purine synthesis occurs in 4 Phases
NOT expected to know the whole pathway, key regulatory steps, modulators
Here showing again the atomistic sources
*Committed step
Formation of inosine monophosphate (IMP) is a branching point
Describe phase I of purine synthesis in detail
•Phase I: activation of ribose 5-phosphate
–Starts with ribose 5-phosphate, which is a byproduct of the oxidative phase of the pentose phosphate pathway
–Converted to “active” form, 5-phosphate-α-D ribosyl 1-pyrophosphate (PRPP)
- Utilizes ATP
- Requires PRPP synthetase
–Allosterically activated by phosphate levels
•Pi levels signal cellular activity due to ATP consumption
–Negatively regulated by levels of purine nucleotides GMP, AMP, and IMP
PRPP is the phosphorylated form of ribosyl
If your body is consuming a lot of ATP and residual levels of inorganic phosphate are elevated,
Then that triggers the further synthesis of purines to compensate for that additional need of ATP
Describe phase II of purine synthesis in detail.
•Phase II: conversion of PRPP into phosphoribosylamine
–Glutamine:phosphoribosyl pyrophosphate amidotransferase substitutes pyrophosphate with an amino group at C-1ʹ or PRPP
- Obtains the amino group from Glutamine
- Generates phosphoribosylamine (PRA)
–Allosterically positively regulated by PRPP levels
–Negatively regulated by the levels of purine nucleotides GMP, AMP and IMP
*****Committed Step in Purine Biosynthesis: irreversible, rate-determining step
- Formation of the phosphoribosyl amine
- Hypoxanthine ribose phosphate = inosine monophosphate
- Not commonly found in DNA or in RNA
- Converted into Guanosine monophosphate (GMP) and Adenosine monophosphate (AMP)
Describe phase III of purine synthesis in detail.
•Phase III: construction of IMP
–Branch point purine ring
–PRA enters a nine-step ring-constructing sequence that produces IMP
–All intermediates are phosphorylated (nucleotides) due to the phosphate group on ribose 5-phosphate
–Consumes ATP (4 eq.) in reaching IMP
–IMP is the branch point in anabolism of purines
–2 C’s from folate derivative, 1 C from CO2, remaining C’s and N’s from amino acids, Gln, Gly and Asp
Describe phase IV of purine synthesis in detail.
•Phase IV: conversion of IMP into dATP and dGTP
–AMP negatively controls adenylosuccinate synthetase
–GMP negatively controls IMP dehydrogenase
–Conversion of IMP to XMP is a oxidation reaction that requires NAD+
–ATP and GTP are used in the synthesis of GMP and AMP, respectively.
–Conversion of IMP to XMP is the rate-limiting step in GTP synthesis
- Conversion of IMP to XMP is a oxidation reaction that requires NAD+
- ATP and GTP are used in the synthesis of GMP and AMP, respectively. Balance between the two pool of primary purines is maintain by consuming one purine nucleotide triphosphate during the synthesis of the other.
- How fumarate gets replenished!
- The formation of PRA is the rate-limiting step of purine synthesis (catalyzed by Glutamine:phosphoribosyl pyrophosphate amidotransferase a.k.a. Amidophosphoribosyl transferase) but both of the first two steps are important regulatory steps. However and more specifically, the rate-limiting step in the de novo synthesis of GTP is the conversion of IMP to XMP by IMP dehydrogenase. This comes into play in the highlighted correlation box where we discuss depriving lymphocytes dGTP and GTP to suppress immune function to help prevent rejection!
How is purine synthesis regulated?
•Feedback Inhibition à accumulation of the end-product inhibits its own synthesis
–Synthesis of PRPP
–Synthesis of phosphoribosyl amine
–Synthesis of AMP and GMP from IMP
•Cross-Regulation:
–AMP synthesis is stimulated by GTP
–GMP synthesis is stimulated by ATP
How does your body know with pathway to upregulate?
Formation of end-products inhibits the pathway.
Don’t want to make too much of A without G, so they are cross-regulated
Describe pyrimadine synthesis.
- Phase I: fabrication of pyrimidine ring as orotate
- Phase II: attachment of orotate to PRPP to generate uridine monophosphate, the branch point pyrimidine ring synthesis
- Phase III: conversion of UMP to CTP and dTMP
Pyrimidine synthesis occurs in 3 phases
The pyrimidine is first synthesized and then the activated ribose is added.
Phosphoribosyl pyrophosphate (PRPP) is a pentosephosphate. It is formed from ribose 5-phosphate by the enzyme PRPP synthetase (aka ribose-phosphate diphosphokinase).
Describe phase I of pyrimadine synthesis in detail.
•Phase I: fabrication of pyrimidine ring as orotate
–Rate-limiting step: formation of carbamoyl phosphate
–Carbamoyl phosphate synthetase II is activated by PRPP and inhibited by UTP
–Defect in the urea cycle can result in elevated levels of carbamoyl phosphate and manifests as hyperammonemia with orotic aciduria
- Ammonia from Gln with bicarb and 2x ATP is condensed to give carbamoyl phosphate via carbamoyl phosphate synthetase II
- Occurs both in the cytosol and the mitochondria
Describe phase II of pyrimadine synthesis in detail.
•Phase II: attachment of orotate to PRPP to generate UMP
–UMP synthetase attaches orotate to PRPP to give orotidine monophosphate (OMP)
–UMP synthetase then decarboxylates OMP to generate UMP
- UMP synthetase is a bifunctional enzyme: two main domains, an orotate phosphoribosyltransferase (OPRTase) and an orotidine-5’-phosphate decarboxylase (ODCase) subunit.
- Catalyze the last two steps UMP synthesis.
- Addition of ribose-P to orotate by OPRTase forms OMP
- OMP is decarboxylated to UMP by ODCase
- Orotic aciduria: inability to convert orotic acid to UMP, causes megaloblastic anemia. Associated with mental and physical developmental delays
- Treated with oral uridine
Describe phase III of pyrimadine synthesis in detail.
•Phase III: conversion of UMP into cytosine and thymidine (deoxy) nucleotides
–UDP acts as a central portal to other pyrimidines
–dUDP loop is wasteful, but thought that this occurs so that dUTPase keeps dUTP low to prevent incorporating into DNA
–dUMP is bridge to thymidine production
–UTP is aminated to form CTP
First reduces UDP to dUDP (deoxy thymidine used in DNA synthesis, exclusively)
Various kinases and phosphatases interconvert dUDP dUTP and dUMP
CTP synthase is an aminotransferase, converts UTP to CTP
- Phosphatase dUTPase in important for genomic regulation
- Wasteful in that is dUDP is first converted to dUTP and then back to dUMP, but dUTPase keeps dUTP levels low so that it doesn’t incorporate into DNA
- dUMP serves as a bridge to thymidine nucleotides as it undergoes methylation for conversion into dTMP
- Methyl groups originates from a folate derivative
- Therapeutic window in that this methylation step can be targeted to rapidly dividing cells
What are the key regulatory steps of pyrimadine synthesis?
•Carbamoyl phosphate synthetase
–Inhibited by UMP/UTP
–Stimulated by PRPP
•Aspartate transcarbamoylase (ATCase)
–Inhibited by CTP
- Phosphatease dUTPase in important for genomic regulation
- Wasteful in that is dUDP is first converted to dUTP and then back to dUMP, but dUTPase keeps dUTP levels low so that it doesn’t incorporate into DNA
- dUMP serves as a bridge to thymidine nucleotides as it undergoes methylation for conversion into dTMP
- Methyl groups originates from a folate derivative
- Therapeutic window in that this methylation step can be targeted to rapidly dividing cells
Summary of de novo Nucleotide Synthesis table
What is the pentose phosphate pathway?
Pentose phosphate pathway (pg. 276): in humans, the pentose phosphate pathway produces ribose 5-phosphate and NADPH.
- In erythrocytes, this occurs in the liver, testes, mammary glands and the adrenal cortex.
- Uses NADPH to maintain a reducing environments (reduced form of glutathione) and to provide reducing power for biosynthesis of fatty acids and steroids.
- Formation of glutathione (GSH) as a key antioxidant in detoxification and reducing oxidative damage
- In humans, liver is principle site of purine and pyrimidine synthesis and utilizes ribose 5-phosphate and 3/5 principle free amino acids in the liver (Asp, Gln, Gly) as starting materials.
Produces ribose sugars
Produces NADPH: production of other things
NADH product of TCA c —> eventual production of ATP (energy)
Cycle and drives ATP synthesis
Glutathione helps body detoxify via a reducing environment, get rid of oxidizing species
What is methotrexate?
Methotrexate (pg. 275): antineoplastic agent used to treat cancer
- Targets dihydrofolate reductase (DHFR), which converts dietary folate to the biologically active tetrahydrofolate in the liver.
- Methotrexate prevents oxidation of NADPH
- Inhibition disrupts DNA replication in rapidly dividing cancer cells.
- Efficacy depends on selective drug uptake by cancer cells compared to normal cells.
- Very similar in structure
- Competitive inhibitor of many enzymes that utilize folate
- Methotrexate Prevents Oxidation of NADPH by DHFR (dihydrofolate reductase)
- Methotrexate binds dihydrofolate reductase 100 fold more tightly
- This inhibition disrupts DNA replication in rapidly dividing cells
- Methotrexate prevents oxidation of NADPH
What is fluorouracil?
[[[[[BLUE BOX]]]]]
Fluorouracil (pg. 275): used to treat cancer, brand name Adrucil. Injected to treat colon, esophageal, breast, cervical, pancreatic cancers; topical use in warts, carcinomas.
- Targets thymidylate synthase essentially stopping DNA synthesis.
- Thymidylate synthase methylates dUMP to dTMP.
- Treatments with 5-FU causes scarcity of dTMP.
- Triggers cell death in rapidly dividing cancer cells.
- Medical use started in 1962, WHO “Essential medicines”
- Anti-cancer agents can block thymidylate synthase and dihydrofolate reductase
- Reacts with methylene-tetrahydrofolate to stable adduct to the enzyme and prevent any catalytic activity
What are ‘sulfa’ drugs
[[[[[BLUE BOX]]]]]
“Sulfa” drugs (pg. 275): antibacterial agents in the sulfonanilamide family (“sulfa”) competitively inhibits the bacterial enzyme that incorporates p-aminobenzoic acid (PABA) into folate
- PABA is an intermediate in the synthesis of folate by bacteria, plants, and fungi
- Sulfa drugs selectively disrupts DNA replication in bacteria
- Humans acquire folate as a vitamin in their diets
- Selective to bacteria
- Taking advantage of the similar folate pathway
- Humans require folate from their diet (essential)
- Bacteria synthesize it themselves (nonessential)
- Inhibiting the folate pathway could be a viable antibacterial application
What happens when you deprive cells of GMP and dGTP?
[[[[[BLUE BOX]]]]]
Depriving cells of GMP and dGTP (pg. 285): oxidative step in conversion of IMP to XMP is an opportunity of therapeutic intervention
- IMP dehydrogenase is the target
- Therapeutic agent is an immunosuppressant that disrupts DNA replication in B and T cells
- Works by depriving them of adequate supplies of GMP and, hence, dGTP
- Disrupting GMP synthesis is useful in preventing rejection, anti-tumor
- Inhibition of IMP dehydrogenase
- De novo synthesis operates in lymphocytes
MMF was approved by the U.S. Food and Drug Administration (FDA) in 1995 as an immunosuppressant drug for use in solid organ transplantation. What is MMF and what are its indications?
MMF- Mycophenolate Mofetil
(see table)
Describe purine catabolism.
- Removal of ribose from guanosine and inosine produces guanine and hypoxanthine
- Converges at formation of xanthine
- Largely converted to uric acid
- Adenosine deaminase (ADA):
–Irreversible hydrolytic deamination
–Adenosine —> inosine
–Overproduction of erythrocyte isoform causes hemolytic anemia
–Underproduction associated with SCID
•Xanthine oxidase:
–Hypoxanthine —> Xanthine —> Uric Acid
–Target for gout treatment
Describe the importance of oxidation levels of purines.
[[[[[CORRELATION BOX]]]]]
Oxidation levels of purines (pg. 277): typically, catabolic processes include steps where oxygen is added to molecules to make them more polar
- Adenosine = 0 Oxygen, guanine and hypoxanthine = 1 Oxygen, xanthine = 2 Oxygen, uric acid = 3 Oxygen
- Uric acid, as the end point of purine catabolism, is the most oxidized
- Has an acid hydrogen and limited solubility in water that plays a key role in gout
What is Gout?
[[[[[BLUE BOX]]]]]
Gout (pg. 278): characterized as high levels of uric acid in the blood.
- Primary hyperuricemia à overproduction of uric acid
- Secondary hyperuricemia à underexcretion of uric acid
- Results in extremely painful deposits of sodium urate in the joints of extremities (gouty arthritis); 3+ million cases a year in U.S.
- Sodium urate deposits in the kidneys can cause damage.
- Diets rich in purines (beans, spinach, lentils) along with alcohol, meat and seafood can trigger episodes.
- Acidemia stimulates reabsorption in the kidney by URAT1.
- Treatment includes reducing the amount of granulocytes to affected areas and allopurinol that inhibits xanthine oxidase
- Also proposed to increase levels of more soluble purines hypoxanthine and guanine
Describe pyrimidine catabolism.
•Converted to readily metabolized ketogenic or glucogenic, water-soluble compounds
–Malonyl coenzyme (CoA), methylmalonyl CoA, and succinyl CoA
–Uracil/cytosine —> malonyl CoA (ketogenic)
–Thymine —> methylmalonyl CoA or succinyl CoA (glucogenic)
Less convergence in pyrimidine catabolism compared to purine
What are salvage pathways?
- Bases recovered during nucleotide turnover or digestion can be reincorporated into nucleotides
- Dominates de novo synthesis for purines
–Adenine phosphoribosyltransferase (APRT) generates AMP
–Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) generates GMP or IMP
What is Acyclovir?
[[[[[BLUE BOX]]]]]
Acyclovir (pg. 285): Thymidine kinase phosphorylates the nucleotide deoxythymidine (dT) to generate dTMP using ATP as the phosphoryl donor.
- The antiviral agent acyclovir (which has a base that more closely resembles guanine than dT), undergoes phosphorylation by viral thymidine kinase at a rate that far exceeds that of cellular kinase.
- Viral thymidine kinase rapidly converts acyclovir to its monophosphate acyclo-dGMP.
- Other kinases then convert acyclo-dGMP to acyclo-dGTP.
- Acyclo-dGTP is incorporated into rapidly dividing viral cells.
- However, the acyclo-dGTP lacks a 3’-OH group which terminated DNA replication
- Acyclovir is used to help heal sores related to chicken pox, shingles and HPV.
- Nucleoside analogue
- Viral thymidine kinase affinity is much increased for acyclovir
What is the mechanism of action of Acyclovir?
see pic
- Nucleoside analogue
- Viral thymidine kinase affinity is much increased for acyclovir
