Mkgrady8 Purine And Pyrimidine Metabolism

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gouty arthritis

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caused by accumulation of a breakdown product of purine nucleotide catabolism - uric acid; metatarsal-phalangeal joint at base of big toe is affected in 50% of cases

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adenosine deaminase deficiency

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the 1st disease in humans to be treated with gene therapy

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nucleotide synthesis and entineoplastic treatments

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several common chemotherapeutic drugs target specific steps or enzymes associated with nucleotide synthesis= glutamine analogs, unique enzymes (thymidylate synthase), unique cofactors (tetrahydrofolate)

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neucleotide nomenclature

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for purine (adenine, and guanine) the ending is -ine if it is just a base and -osine if you add the base to it; for pyrimidines (cytosine and uracil/thymine) the ending is -ine if it is just the base and -idine if you add the base to it; for either if you have all of the phosphates on it then the ending is -ylate

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numbering system for the purines (R)

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atom numbering is counter-clockwise and they are jointed to the sugar moiety through N-9

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numbering system for pyrimidines (Y)

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atom numbering is clockwise and they are joined to the sugar moiety through N-1

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draw out the purine ring system and think about what would make it each of the purines

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draw out the pyridine ring structure and then think about what would make it each of the pyrimidines

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other purines not found in nucleotides

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caffeine- plant product most commonly consumed from coffee or tea plants, mild stimulant in humans, inhibitor of acetylcholinesterase; theobromine- aka xantheose from cacoa plant, found in chocolate, acts as a vasodilator, diuretic, and heart stimulant; theophylline- found in tea leaves and cocoa beans, used to treat respiratory diseases such as COPD and asthma

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sugar components of nucleotides

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ribose and its derivative deoxyribose are products of the pentose phosphate pathway; numbering uses prime indicator to differentiate positions on sugars and bases; bases are joined to sugars through beta-gycosidic linkages at the 1' position of the sugar

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draw out ribose and show a beta glycosidic linkage

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draw out deoxyribose and show a beta-glycosidic linkage

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sources of nucleotides

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ribonucleotides- cellular concentration from food in millimolar range; deoxyribonucleotides- micromolar range; dietary- minimal, most degraded in intestinal mucosa; thus cells rely on the synthesis of nucleotides- both de novo and salvage pathways, de novo synthesis rates differ between cell types and developmental stage, liver cells are primary source of nucleotides in adults while nucleotide synthesis levels are extremely low in neural cells

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nucleotide biosynthesis- salvage pathway

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activated ribose (PRPP)+base --> nucleotide; salvage pathways exist for both purines and pyrimidines; the purine salvage pathways are especially important for energy saving and effects of their absence; some tissues (most notably neural tissue) rely almost exclusively on salvage pathways

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nucleotide biosynthesis- de novo pathway

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activated ribose (PRPP)+amino acids+ATP+CO2+... -->nucleotide; major difference between purine and pyrimidine biosynthesis is that purine nucleotides assembled on ribose and pyrimidine bases are pre-assembled and then attached to ribose

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formation of PRPP

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purines are assembled stepwise on C-1' of ribose-5-phosphate which is first activated to form phosphoribosyl pyrophosphate (PRPP); transfer to pyrophosphate from ATP to ribose-5-phosphate is catalyzed by PRPP synthetase (very important enzyme-REMEMBER) and this is the rate limiting step of nucleotide biosynthesis; note:rate limiting steps of biochemical pathways are important steps for regulation (e.g. feedback inhibition) so disruption of regulation of this step is of critical importance for understanding the medical complications associated with gouty arthritis

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purine biosynthesis

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purine rings are assembled from glycine, glutamine, aspartate, N10-formyl-tetrahydrofolate and CO2 (or bicarbonate); insinuate (IMP) is the first purine nucleotide constructed and adenine and guanine nucleotides are derived from insinuate; the purine base in IMP is named hypoxanthine; once PRPP is converted to phosphoribosylamine (addition of NH2) you are committed to making a purine; you use a lot of glutamine in this process (so if you replace the glutamine with something similar that prevents production of the purines you can slow down cancers); to make AMP use GTP and to make GMP use ATP (balances out production so they are equal)

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formation of nucleotide di- and triphosphates

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ATP is the source of phosphates for formation of di- and triphosphate forms of nucleotides; specific enzymes are required for the formation of each diphosphate termed nucleotide monophosphate kinases- 2ADP<-->AMP+ATP by adenylate kinase and GDP+ADP<-->GMP+ATP by guanylate kinase; a single enzyme with broad specificity nucleotide diphosphate kinase catalyses the second reaction GTP+ADP<-->GDP+ATP by nucleotide diphosphate kinase

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regulation of purine biosynthesis

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feedback inhibition of de novo purine biosynthesis by nucleotides; PRPP syntheses and glutamine phosphoribosyl aminotransferase inhibited by purine nucleotides and synergistically by AMP and GMP; pathways leading from IMP are inhibited by products of that branch; the reciprocal substrate relation whereby ATP is used in the synthesis of GMP and GTP is used in the synthesis of AMP balances the production of these nucleotides; feedback inhibition is also featured in pyrimidine biosynthesis

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salvage pathways

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regenerate nucleotides from free bases by attaching them to PRPP; energy efficient (no need to reassemble ring structures); hypoxanthine-guanine phosphoribosyl transferase (HGPRT)(very important enzyme to remember) catalyzes the recovery of hypoxanthine or guanine; adenine phosphoribosyltransferase catalyzes the recovery of adenine however most adenine is recovered via inosine; pyrimidine phosphoribosyl transferase catalyzes the recovery or uracil but not cytosine; one step pathway (more efficient)

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hyperuricemia

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uric acid is the end product of purine degradation in humans must be excreted in the urine; uric acid solubility is dependent on several factors including pH and temp; normal uric acid levels in humans are close to its solubility level (7 mg/dL in plasma and interstitial fluids); normal range in males 2-7.5 and females 2-7; uric acid levels increase with age and with weight; elevations in uric acid levels above its solubility level (hyperuricemia) can lead to deposition of uric acid crystals in the urine (kidney stones) or of sodium rate in plasma or interstitial fluids (gout); gouty arthritis is an inflammatory disease caused by NA urate deposition in joints particularly in the periphery where temps are lower

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diagnosis of hyperuricemia

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detection of negatively birefringent crystals under polarized light microscopy in aspirate from affected joint; detection of typhus (deposit of monosodium rate crystals) under skin often on effected joint; blood test (limited utility as uric acid levels can vary in the course of a day)

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causes of hyperuricemia

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anything that causes increases in purine biosynthesis, degradation, or decreases uric acid excretion; metabolic diseases e.g. increased supply of ribose-5-phosphate; increased cell turnover presumably because prunes from these cells must be degraded (e.g. cancer puts undergoing chemotherapy); decreased renal excretion (accounts for approximately 90% of cases); genetic causes; evolutionary advantage?

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what are some genetic causes

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increased PRPP synthase activity (normally rate-limiting); decreased HGPRT activity (salvage enzyme)- partial deficiency leads to Lesch-Nyhan syndrome which is characterized by spasticity, mental retardation, choreoathetosis, and self-mutilation, although individuals are hyperuricemic the neurological symptoms are not caused by uric acid but due to the reliance of the brain on the salvage pathway for purine synthesis; glucose metabolism disorders like von Gierke Disease (results from increased activity of the pentose phosphate pathway)

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treatments for hyperuricemia and gout

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anti-inflammatory drugs- colchicine (but can also cause GI side effects) leads to cell death (more purines to degrade?), NSAIDSs (e.g. indomethacin, ibuprofen); uricosuric agents- increase renal excretion of uric acid; inhibition of xanthine oxidase- allopurinol leads to decreased levels of uric acid and increased levels of hypoxanthine and xanthine, more soluble

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allopurinol mechanism of action

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alloxanthine binds tightly and non-covalently to xanthine oxidase and inhibits its activity; approx 3=5% of pts will develop insensitivity to allopurinol; a related XO inhibitor, oxipurinol (a metabolite of allopurinol) is currently in phase III clinical trials

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treating gout- acute attack (no previous history, sudden onset)

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treat major symptom (pain) so NSAID or colchicine (if they are allergic to NSAID)

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treating gout- chronic condition

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treat underlying condition (e.g. renal disorder) so give uric odic agent allopurinol; tell them to avoid purine rich foods

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lessons learned from gout case

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disease of nucleotide metabolism (excess purine cannot be stored and must be degraded); pt was quite young (affected individuals are more commonly older males, uric acid levels are higher in males and increase with age); dietary induced condition (purines in steak and beer contributed to the development of an acute gout attack); uric acid concentrations are normally close to solubility limit, thus relatively small variations in concentration can trigger onset of symptoms: in acute cases treatment is purely sympomatic; in chronic cases ongoing treatment will be required to decrease uric acid concentrations

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overview of pyrimidine biosynthesis

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the pyrimidine ring is assembled from glutamine (as a source of ammonia), aspartate, and CO2 (or bicarbonate); the initial step involves the synthesis of C-2 and N-3, the other 4 atoms are derived from aspartate; PRPP is again used but is added after ring assembly; UMP is the first pyrimidine nucleotide; VIDEO: need aspartate and carbamoyl phosphate to assemble ring, then add sugar using PRPP to form UTP which them forms the other pyrimidines, UMP synthase carry out the final 2 steps so mutations in this cause a build up in the substrate which is called orotate )hereditary orotic acuduria)

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hereditary orotic aciduria

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caused by mutations that affect the last 2 steps of pyrimidine formation (so mutation in UMP synthetase); orotic acid (orotate) crystals (asymptomatic) in urine of newborns; condition due to deficiency of pyrimidines and can be treated with oral administration of pyrimidines

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UMP to UTP

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UMP+ATP<--> UDP+ADP (UMP kinase (specific)); UDP+ATP<-->UTP+ADP (nucleotide diphosphate kinase (broad range))

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CTP is formed by

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amination of UTP; glutamine is the source of the amine group and the reaction proceeds via activation of C-4 by phosphorylation and replacement with the amine

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formation of deoxynucleotides

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this is the weird way that TTP is formed; formed by the replacement of the hydroxyl group at the 2' position or ribose with a hydrogen ion; catalyzed by riboculeotide reductase (very important enzyme); ribonucleotide reductase activity is highest just prior to S-phase in the cell cycle (i.e. prior to DNA synthesis); ribonucleotide reductase activity is intricately regulated by its products (dATP (THE ULTIMATE SENSOR) negatively regulates its activity (i.e. serves as a general sensor of deoxyribonucleotide levels) and the other dNTPs modulate substrate specificity to ensure a balanced production of each dNTP)

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formation of deoxynucleotides continued

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replace 2' OH with H

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regulation of ribonucleotide reductase

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expression of RR is controlled at the level of the cell cycle (increases prior to DNA synthesis); dATP levels control the overall activity of RR (if dATP levels are high RR activity is decreased)(note relevance to ADA deficiency); levels of dCTP, dGTP, and TTP control substrate selectivity of RR

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thymidylate is formed by

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methylation of deoxyuridylate; methyltetrahydrofolate is converted to dihydrofolate during this reaction; the tetrahydrofolate must be regenerated to permit the formation of more TTP (this is catalyzed by dihydrofolate reductase (DHFR) (remember this enzyme)); dUMP to dTMP using the enzyme thymidylate synthase (remember this enzyme); the remember enzymes here are major targets of cancer therapy because dTTP is important in forming DNA and you don't want cancer cells to divide

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dUMP is formed from

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dUDP via dUTP (cleaved by dUTPase to form dUMP and PPi); alternatively dUMP can be formed by deamination of dCMP

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degradation of pyrimidines

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become water soluble and are released in urine

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adenosine deaminase deficiency

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deficiency in adenosine deaminase; signs and symptoms= SCID

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purine nucleoside phosphorylase deficiency

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deficiency in purine nucleoside phosphoylase; signs and symptoms= immunodeficiency with T cell defect

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familial orotic aciduria

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deficiency in orotate phosphoribosyl-transferase; signs and symptoms= accumulation of orotic acid in blood and urine, failure to thrive

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Lesch-Nyhan syndrome

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deficiency in HGPRT; signs and symptoms= mental retardation with self mutilation, hyperuricemia

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ADA deficiency

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one form of severe combined immunodeficiency (SCID) is caused by mutations that block ADA production; results in a lack of functional T and B cells; due to a build up in the levels of dAMP, dADP, and dATP; inhibition of ribonucleotide reductase by dATP is implicated in the immunodeficiency thereby blocking DNA synthesis; reason for cell selectivity is unknown; another form results from purine nucleoside phosphorylase deficiency which results from purine nucleoside phosphorylase deficiency which results from defects in T cell function but the mechanisms are currently unknown

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cancer treatments

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cancer cells are characterized by increased cell division therefore deprivation of nucleotides can be used as a treatment for cancer; glutamine analogs inhibit steps that use glutamine as a source for ammonia (e.g. azaserine); structural analogous of bases or nucleosides either inhibit specific enzymes or disrupt DNA or RNA structure (e.g. flurouracil= suicide inhibitor (covalently attaches and takes it out of commission) of thymidylate synthase); antifolates- methotrexate and aminopterin (analogs of dihydrofolate) block thymidylate synthesis by inhibiting DHFR activity leading to depletion of tetrahydrofolate

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nucleotide modifications

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chemical modifications of nucleotides occurs in messender RNAs (addition of methyl guanine cap at 5' end) and transfer RNAs (several bases may be methylated); deoxyribonucleotides may also be modified in DNA in both prokaryotes and eukaryotes; in prokaryotes base methylation protects DNA from digestion by restriction enzymes and functions as a kind of primitive immune system; in eukaryotes only on base (cystine) can be modified by methylation and this modification always occurs in the context of CpG dinucleotide

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consequences of DNA methylation in eukaryotes

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alteration of chromatin structure; alteration of binding of proteins to DNA; alteration of gene expression patterns

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DNA methylation at the genome level

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chromosomes are globally demethylated during gametogenesis; chromosomes are remethylated during embryogenesis; hypermethylated regions of DNA are organized into heterochromatin (generally unexpressed regions of chromosomes); hypomethylated regions of DNA are organized into euchromatin (generally contains genes that are being expressed (or can be expressed))

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biological relevance of DNA methylation

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alteration of gene expression (most commonly inhibition of expression) (ex loss of expression of tumor suppressor gene expression in cancer); alteration of chromosome structure (ex inactivation of one X chromosome in the cells of female humans); inheritance of gene expression patterns in the absence of changes in DNA sequence (i.e. mutations)(ex epigenetic inheritance in Genomic imprinting); inhibition of gene expansion in somatic cells (ex FMR1 gene in Fragile X Syndrome)

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summary: nucleotides can be synthesized how

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de novo or recycled by salvage pathways

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summary: purine rings are assembled how

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on a ribose support while pyrimidine rings are synthesized prior to attachment of ribose

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summary: deoxynucleotides are synthesized how

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by reduction of ribonucleotides

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summary: nucleotide biosynthesis is regulated by

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feedback inhibition of key synthetic steps

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summary: nucleotides are important constituents of

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several cofactors and other biomolecules

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summary: disruptions in nucleotide metabolism underlie what

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several human disorders and diseases including hyperuricinema, Lesch-Nyhan disease, and some forms of immunodeficiency

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summary: cancer treatments often target what

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nucleotide biosynthetic pathways as a means of specifically targeting rapidly dividing cells

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starting here from video

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yes

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don't need to know numbering of rings of purine and pyrimidines except the number of the locations of the sugar addition; so what are they

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for purines: sugar joined at N-9; for pyrimidines sugar joined at N-1

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don't need to know the structure of the nucleic acids but should know how they differ; so how do they differ

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addenine and guanine are 2 ring systems and have NH2 and =O respectively; cytosine, uracil, and thymine are 1 ring systems and have NH2, =O, and =O with CH3 respectively

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the intermediate that then goes on to form adenine or guanine

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hypoxanthine

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cells cannot store what

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purine molecules

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very important enzyme xanthine oxidase

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converts hypoxanthine to xanthine and then to uric acid; the best treatment for gout is an inhibitor of xanthine oxidase