Nucleotide Metabolism Flashcards
What are the two types of heterolytic rings?
Purines and pyrimidine
What are nucleotides?
The phosphate esters of nucleosides
Nitrogenous base + sugar + phosphate
What are nucleosides composed of?
Nitrogenous base + sugar
True or false: dATP is equivalent to ATP
False
What is different about a deoxynucleotide?
There is no OH group on C2
Hypoxanthine and xanthine are classified as purine or pyrimidines?
Purines
What are the two ways in which purines can be synthesized?
De novo synthesis and salvage pathway
Briefly describe de novo purine synthesis and its location
Site: liver, cytosol
Formation of purine base on ribose-5-phosphate from the PPP
Briefly describe the purine salvage pathway and its location
Site: organelles
Addition of ribose 5P to the preformed purine base
What are the two ways in which pyrimidine nucleotide synthesis can occur?
De novo synthesis and the salvage pathway
Briefly describe de novo pyrimidine synthesis and its location
Site: liver, cytosol, mitochondria
Formation of pyrimidine ring structure followed by the addition of ribose phosphate
Briefly describe the pyrimidine salvage pathway and its location
Site: organelles
Formation of pyrimidine nucleotides from pyrimidine bases in RNA/DNA
What are the phases of purine synthesis?
- Activation of ribose 5 phosphate
- Conversion of PRPP into phosphoribosylamine
- Construction of inosine monophosphate (branch point)
- Conversion of IMP into adenosine and guanosine
Describe phase I: activation of ribose 5 phosphate in purine synthesis
Starts with ribose 5 P which is a byproduct of the oxidative phase of the PPP
Converted to active form, 5-phosphate alpha D ribosyl - 1-pyrophosphate (PRPP)
Utilizes ATP and requires PRPP synthetase
Explain the specific roles of nucleosides and nucleotides
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-Gln and NADPH and NADH
Nucleotides such as cAMP and cGMP play regulatory roles and serve as stabilizing energy elements
Nucleosides also appear in important biomolecules such as adenosine in vitamin B12
What is PRPP synthetase (phase I purine synthesis) regulated by?
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
Describe phase II: conversion of PRPP into phosphoribosylamine in purine synthesis
Glutamine:phosphoribosyl pyrophospphate aminotransferase substitutes pyrophosphate with an amino group at C1 or PRPP
Obtains the amino group from glutamine
Generates phosphoribosylamine (PRA)
What is phase II of purine synthesis regulated by?
Allosterically positively regulated by PRPP levels
Negatively regulated by the levels of purine nucleotides GMP, AMP and IMP
Describe phase III: construction of inosine monophosphate in purine synthesis
PRA enters 9 step ring constructing sequence that produces IMP All intermediates are phosphorylated (nucleotides) due to the phosphate group on R5P Consumes ATP(4) in reaching IMP IMP is the branch point in anabolism of purines
Where do the C and N come from in purine synthesis?
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: conversion of IMP into adenosine and guanosine in purine synthesis
Conversion of IMP to XMP is an 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 RLS in GTP synthesis
Describe the feedback inhibition that occurs in purine synthesis
Synthesis of PRPP, phosphoribosyl amine, AMP and GMP from IMP
Accumulation of these end products inhibit their own synthesis
Describe the cross regulation that occurs in purine synthesis
AMP synthesis is stimulated by GTP
GMP synthesis is stimulated by ATP
What are the phases of pyrimidine synthesis?
- Formation of pyrimidine ring as orotate
- Attachment of orotate to PRPP to generate uridine monophosphate (branch point)
- Conversion of uridine monophosphate into cytosine and thymidine (deoxy) nucleotides
Describe phase I: formation of pyrimidine ring as orotate in pyrimidine synthesis
RLS: formation of carbamoyl phosphate
Last step that generates orotate is in the mitochondria, then orotate is transported to the cytosol
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 manifest as hyperammonia with orotic aciduria
Describe phase II: attachment of orotate to PRPP to generate uridine monophosphate (the branch point of pyrimidine synthesis)
UMP synthetase attaches orotate to PRPP to give orotidine monophosphate (OMP)
UMP synthetase then decaroxylates OMP to generate UMP
Describe phase III: conversion of uridine monophosphate into cytosine and thymidine deoxynucleotides
UDP acts as a central portal to other pyrimidines
dUDP loop is wasteful but through 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
Describe the key regulatory steps of pyrimidine synthesis
Carbamoyl phosphate synthetase is inhibited by UMP/UTP and stimulated by PRPP Aspartate transcarbamoylase (ATCase) is inhibited by CTP
Why is the PPP important?
In humans the PPP produces R5P and NADPH NADPH used to maintain reducing environments and to provide reducing power for biosynthesis of FAs
Formation of glutathione as a key antioxidant
Liver is principle site of purine and pyrimidine synthesis and utilizes R5P and 3/5 principle free amino acids in the liver (Asp, Gln, Gly) as starting materials
What is the difference between the source of atoms for C H O N in purines and pyrimidines?
Both use Gln, Asp, CO2 and N-formyl THF
Pyrimidine synthesis does not use Gly whereas purines do
What is methotrexate?
Anti-neoplastic agent used to treat cancer
Targets dihydrofolate reductase (DHFR) which converts dietary folate to the biologically active tetrahydrofolate in the liver
Prevents oxidation of NADPH
Inhibition disrupts DNA replication in rapidly dividing cancer cells
What is fluorouracil?
Cancer treatment drug
Inhibits thymidine synthase and therefore inhibits thymidine pathway (dUMP -> dTMP)
What are sulfa drugs?
Antibacterial agents in the sulfa family that competitively inhibit 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 disrupt DNA replication in bacteria
Humans require folate as a vitamin in their diet and do not make it so these drugs are selective to bacteria
Describe the method of depriving cells of GMP and dGTP
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 disputes 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 transplant rejection
Describe purine catabolism
Removal of ribose from guanosine and inosine produces guanine and hypoxanthine
Converges at formation of xanthine
Largely converted to uric acid
Describe the role adenosine deaminase (ADA) plays in purine catabolism
Irreversible hydrolytic deamination
Adenosine —> insosine
Overproduction of erythrocyte isoform causes hemolytic anemia
Underproduction associated with SCID
Describe the role xanthine oxidase plays in purine catabolism
Hypoxanthine —> xanthine —> uric acid
Target for gout tx
Describe the oxidation levels of purines
Typically catabolic processes include steps where oxygen is added to molecules to make them more polar Adenine = 0, guanine and hypoxanthine = 1 oxygen, xanthine = 2 oxygens and uric aid =3 oxygens Uric acid (the end point of catabolism) is the most oxidized and has an acidic hydrogen which makes it have limited solubility in water that plays a key role in gout
Describe how uric acid levels are used as a diagnostic marker for gout
Serum uric acid levels are diagnostic marker for gout
Adult males 4-8.6mg/dL and females 3-5.9
Urinary urate levels are normally less than 750mg/24hr
Serum urate levels >9 increase the risk of gout
Urinary urate levels are variable day to day and are the most reliable when the patients are on a low purine diet
What is gout?
Characterized as high levels of uric acid in the blood
Primary hyperuricemia —> overproduction of uric acid
Secondary hyperuricemia —> underexcretion of uric acid
Results in painful deposits of Na urate in the joints of extremities (gouty arthritis)
Na urate deposits in the kidneys can also cause damage
Diets rich in purines (beans, spinach, lentils) along with alcohol, meat and seafood can trigger episodes
Tx 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
What is severe immunodeficiency (SCID)?
Fatal genetic disorder in which B and T cells are compromised
Often males —> most common form is X linked
Characterized by early onset (w/in first 3 months of life) of failure to thrive, chronic diarrhea, thrush and recurrent viral, bacterial, fungal and protozoal infection
Mutations to the receptors shared by ILs involved in development and differentiation of B and T cells
What is ADA deficiency?
The second most common form of SCID (most pronounced in lymphocytes with high ADA activity)
Leads to increased adenosine and decreased inosine
Adenosine subsequently converted to AMP and ADP and then to dADP and dATP
Increased dATP inhibits the activity site of ribonucleotide reductases that in turn blocks the formation of all other dNDPs
Decreased dNDP and dNTP impairs DNA synthesis and leads to the compromised immune system
Describe pyrimidine catabolism
Converted to readily metabolized ketogenic or glucogenic water soluble compounds
Malonyl CoA, methylmalonyl CoA and succinyl CoA
Uracil/cytosine —> malonyl CoA (ketogenic)
Thymine —> methyl malonyl CoA or succinyl CoA (glucogenic)
Describe nucleotide salvage pathways
Bases recovered during nucleotide turn over or digestion can be reincorporated into nucleotides
Dominates de novo synthesis for purines
Adenosine phosphoribosyltransferase (APRT) generates AMP
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) generates GMP or IMP
Patients with <1.5% normal HGPRT activity
LNS presents with additional severe neurologic problems including spastic cerebral palsy, choreoathetosis and self destructive biting (fingers and lips)
Patients with >8% normal HGPRT activity
Kelley-Seegmiller syndrome results with gout and kidney destruction without neurologic sx
Patients with 8-15% normal HGPRT activity
Results in variant LNS with neurologic problems ranging from clumsiness to motor dysfunction
Allopurinol can reduce joint and kidney problems but has not effect on neurological ones
What is Lesch-Nyan syndrome?
Results from defects in HGPRT in purine salvage pathway and is a rare form of primary hyperuricemia
Hyperuricemia leads to gout, urate kidney stones, poor muscle control, mental retardation and tendency for self mutilation
Normally levels of IMP and GMP are maintained in synthesis from PRPP and the salvage pathway mediated by HGPRT
Excess purines are processed by nucleosidases and nucleotidases that convert GMP and IMP to guanosine and inosine and ultimately to uric acid
What do defects in the salvage pathway lead to in LNS?
Excess guanine and hypoxanthine not used in salvage pathway are shunted to form 6x normal levels of uric acid
Purine biosynthesis proceeds at levels 200x normal and underlies the mental retardation and self mutilation
PRPP which is not used in salvage pathway is available for additional purine biosynthesis and allosterically activates the next enzyme in purine biosynthesis
Additional PRPP leading to more PRA has mass action effect on additional synthesis for more purines
What is acyclovir?
Thymidine kinase phosphorylates the nucleotide deoxythymidine (dT) to generate dTMP using ATP as the phosphoryl donor
The antiviral agent that has a base resembling guanine undergoes phosphorylation by viral thymidine kinase at a rate that far exceeds that of cellular kinase
This rapidly converts acyclovir to its monophosphate acyclo-dGMP which then gets converted to acyclo-dGTP and incorporated into rapidly dividing viral cells
Acyclovir is used to help heal sores related to chicken pox, shingles and HPV
