Flashcards in Unit 6 - Nucleotide metabolism Deck (48):
what are the purines?
what are the pyrimidines?
what is PRPP? how is it formed? what is it required in?
-intermediate of major significance in nucleotide metabolism
-made from ribose-5-phosphate and ATP, catalyzed by PRPP synthetase
-required in de novo synthesis and salvate pathways of nucleotides, as well as biosynthesis of nucleotide coenzymes (NAD, FAD)
what are the ways to get ribose-5-phosphate for PRPP?
-glucose metabolism (pentose phosphate shunt)
-nucleoside degradation makes ribose-1-P, which can be converted to R5P
what are the origins of carbons and nitrogens of purine rings?
base: built on molecule of PRPP
ring precursors: gln, gly, CO2, asp, and 2 one-C fragments from one-C folate pool
how is inosine 5'-monophosphate made? what is needed? what are important things to remember?
ten-step process that uses 6 high-E phsophate bonds (energetically expensive; usually salvage 90% of nucleotides, or get from diet)
1. first step (PRPP synthetase) is rate limiting and regulated step
2. two steps need folate, and are blocked by drugs that block folate biosynthesis in bacteria
3. two steps need gln aminotransferase reactions inhibited by azaserine
how does azaserine affect IMP synthesis?
blocks amide transfer from gln (analog of gln)
how do sulfonamides affect IMP synthesis?
blocks biosynthesis of folic acid in bacteria and prevents its formation to block nt synthesis
-analogs of p-aminobenzoic acid
how are AMP and GMP made from IMP? how much energy is needed?
adenylosuccinate made by adding asp to IMP
-fumarate cleaved to make AMP
xanthosine MP made by oxidation of IMP
-amino group from gln is added to make GMP
GTP is cleaved to make AMP, and ATP is cleaaved to make GMP to balance adenine and guanine levels
how is de novo synthesis of purine nucleotides regulated?
-enzymes that catalyze first 2 steps of IMP synthesis (PRPP synthetase and PRPP amidotransferase) both blockedinhibited by IMP, GMP, and AMP
--PRPP amidotransferase has 2 allosteric sites (one for IMP or GMP, the other for AMP), and if both occupied, inhibition is synergistic
-AMP blocks adenylosuccinate synthesis
-GMP blocks XMP synthesis
-substrate channeling and intracellular localization w/in purinosome
what happens in the salvage pathway for purine nucleotides?
uses 2 ribose phosphate transfer enzymes
1. hypoxanthine-guanine phosphoribosyl transferase (HGPRT) to make nt (IMP or GMP) from hypoxanthine or guanine and PRPP
-inhibited by IMP/GMP
2. adenine phosphoribosyl transferase (APRT) makes AMP from adenine and PRPP
-inhibited by AMP
3. nucleoside kinase is only in humans and not really used b/c makes AMP + ADP from adenosine + ATP
how much of free purine is salvaged and recycled?
what are purinosomes?
human-specific organelle where all purine biosynthetic machinery localizes
-presence is regulated by purine abundance
-all 10 steps catalyzed by 6 different enzymes localize here, along with purine salvage pathway enzymes
origins of C and N in pyrimidine rings
precursors: carbamoylphosphate and aspartate
base: pyrimidine ring is formed, then reacts with PRPP to form nucleotide
steps in formation of uridine 5'-monophosphate (uridylate; UMP)
1. carbamoyl phosphate is made in cytosol from gln + CO2, or liver mitochondria as intermediate of urea in synthesis (use different enzymes)
2. another enzyme converts CP to orotic acid in 3 step process that needs asp and NAD+
3. next 2 steps catalyzed by a different enzyme that makes UMP from orotic acid + PRPP (and releases CO2 and PPi)
why do patients with OTC deficiency build up high levels of pyrimidines?
they build up excess carbamoylphosphate (that can't enter urea cycle), and they are instead converted into pyrimidines
what is orotic aciduria? how is it treated?
genetic disorder of pyrimidine biosynthesis
-orotic acid accumulates in blood and is excreted in urine
-alleviated by feeding uridine or cytidine to supply body with needed pyrimidine bases and inhibit CPII via negative inhibition
how is UMP converted to UTP? what enzymes are used?
1. UMP kinase (specific): UMP + ATP --> UDP + ADP
2. nucleoside diphosphate kinase (broad specificity): UDP + ATP --> UTP + ADP
how to make CTP from UTP?
gln aminotransferase reaction
-amino group from gln is donated to UTP to make CTP
how is pyrimidine synthesis regulated?
not well understood
1. enzymes: single PRO consists of 3 identical subunits that catalyze first 3 steps of synthesis, while last 2 steps are catalyzed by another multifunctional enzyme
2. pyrimidine nucleotides decrease asp transcarbomoylase and dihydroorotase activities in multifunctional enzyme, but unknown basis for activity
what are the free pyrimidine bases from nt degradation?
uracil and thymine
how is uracil converted to UMP?
1. uridine phosphorylase: uracil + R1P --> uridine + Pi (reverse of degradation)
2. uridine kinase: uridine + ATP --> UMP + ADP
how is CMP made?
cytidine is phosphorylated by uridine kinase
what are the enzymes that use gln as an NH3 donor, and what are they all inhibited by? what reactions do they progress?
all inhibited by azaserine
1. CPSII - carbamoyl phosphate synthase II (in pyramidine synthesis)
2. CTP synthase - rate-limiting enzyme for synthesis of cytosine nucleotides from both de novo and uridine salvage pathways
3. FGAR aminotransferase
4. GMP synthase - purine synthesis
5. PRPP aminotransferase - committing step in de novo purine synthesis
6. asn synthase - produces asn, AMP, glu, and PPi from asp, gln, and ATP
how often are deoxyribonucleotides made?
cellular levels of deoxyribonucleotides are usualloy low, but increase at time of DNA replication
-99.9% of cells in body are not making them; only cells that are actively dividing and in S phase make them
what are deoxyribonucleotides formed from? what does this mean? what enzyme catalyzes this? what inhibits it?
by reduction of ribonucleoside diphosphates, and ONLY the diphosphates
-this helps prevent dUTP from being formed
-catalyzed by ribonucleotide reductase
--inhibited by hydroxyurea (potent, cheap chemotherapy agent)
how is deoxyribonucleotide formation regulated?
-ribo- and deoxyribonucleotides in a complex fashion that ensures proper ratios and levels of deoxyribonucleotides are present for synthesis of DNA
what are the products of ribonucleotide reductase?
dADP, dCDP, dGDP, and dUDP (which become respective dNTPs)
-since cells don't incorporate dUDP into DNA, it's dephosphorylated to dUMP on its way to become dTTP
how is dTMP formed from dUMP?
catalyzed by thymidylate synthase
-methylene group from N5,N10-methylenetetrahydrafolate is transferred and reduced to methyl group to make dihydrofolate
-THF is regenerated by by dihydrofolate reductase that needs NADPH
--methyl comes from ser --> gly conversion, so one-C groups can be funneled right out of glycolysis and onto dUMP to make dTMP
-carefully regulated in cell cycle dependent manner, and only made by cells in S phase
action of 5-fluorouracil
-how does it mechanicistically inhibit dTMP synthesis?
anticancer agent that undergoes conversion to nucleoside 5'-monophosphate F-UMP in cells
-F-UMP can be phosphorylated to nucleoside diphosphate, and reduced to deoxyribonucleotide, which is dephosphorylated to F-dUMP (fluorodeoxyuridine 5'-monophosphate) that is critical for antineoplastic activity
-F-dUMP interacts with thymidylate synthase and N5,N10 methylenetetrahydrofolate, forming a complex that resembles transition state formed during dUMP --> dTMP conversion
--fluorine atom blocks transfer of methylene group to pyrimidine, so thymidylate synthase is trapped in complex with F-dUMP, so dTMP formation inhibited
action of methotrexate
anticancer analog of folic acid that inhibits dihydrofolate reductase
-regeneration of THF is blocked, and synthesis of dTMP is inhibited
salvage pathway for thymine deoxyribonucleotides
-thymine phosphorylase converts thymine to thymidine (thymine + deoxyribose-1-P --> thymidine + Pi)
-thymidine kinase converts thymidine to dTMP (thymidine + ATP --> dTMP + ADP)
what do nucleotidases do? what are the 4+ types of reactions they catalyze?
remove 5'-phosphates from purine and pyrimidine ribo- and deoxyribonucleotides, converting them to ribo- and deoxyribonucleosides
-AMP: either deaminated to IMP, then hydrolyzed to inosine; or hydrolyzed to adenosine, then deaminated to inosine
-GMP: hydrolyzed to guanosine
-CMP: either deaminated to UMP, then hydrolyzed to uridine; or hydrolyzed to cytidine, then deaminated to uridine
-UMP: hydrolyzed to uridine
what do nucleoside phosphorylases do?
catalyze phosphorolysis of nucleotides to free bases and R1P or deoxy-R1P
-in phosphorolysis, a bond is cleaved by addition of Pi across it to separate ring from ribose (inosine to hypoxanthine; GUT)
-readily catalyze reverse reaction, which is important in salvage pathways, especially of uracil and thymine
what is the final product of purine degradation? how is it made? what molecules are required?
-hypoxanthine from AMP breakdown is oxidized to xanthine (xanthing oxidase), while guanine is deaminated to xanthine
-xanthine is oxidized to uric acid by xanthine oxidase
--needs O2 (to make superoxide, which is converted to H2O2 by superoxide dismutase)
-has molybdenum (only known use), Fe, and S
what form of uric acid is excreted?
enol form is excreted by primates, dogs, birds, and reptiles
what are the 4 general steps that must happen in purine degradation?
3. separation of base from ribose
4. oxidation to uric acid (not specifically in any order)
what is the final product of pyrimidine degradation? what are the 4 general steps that must happen?
beta-alanine (from uracil) and beta-aminoisobutyrate (from thymine)
-must undergo dephosphorylation, separation of base from ribose, deamination, and degradation of base
what is beta-aminoisobutyric acid used as?
marker for cancer cell death, b/c breakdown product of thymine
-can be transaminated to mmCoA
what is gout caused by?
precipitation of sodium urate crystals in joints and kidneys
-crystals precipitate b/c serum levels of urate exceed its solubility limit (elevated uric acid levels may be due to several disorders)
-PRPP synthetase may be abnormal and not responsive to feedback inhibition by purine nucleoside diphosphates
-partial deficiency of HGPRT increases cellular levels of PRPP, which increases de novo synthesis of purines
-no neurologic symptoms
what is the effect of gout?
inflammation and erosion of joints occur when leukocytes engulf deposited crystals, then rupture, releasing lysosomal enzymes
-sodium urate crystals in urinary tract impair renal functions
--in kidneys, causes flank pain
--in extremeities, it starts in big toe
what is treatment of gout? how does it work?
allopurinol (and also colchicine)
-blocks production of uric acid
-allopurinol is oxidized by xanthine oxidase to oxypurinol, which binds to XO and inhibits ability to oxidize xanthing to hypoxanthine (suicide inhibition)
why is allopurinol often given as chemotherapy pretreatment?
during chemotherapy, large quantities of nucleotides are released from killed cancer cells
-this causes uricemia and gout
what is Lesch-Nyhan syndrome caused by?
X-LR due to severe/complete deficiency of HGPRT activity
-increased synthesis of purines, b/c hypoxanthing and guanine not salvaged
-increased intracellular PRPP, and decreased IMP and GMP, causing increased de nova synthesis of purines
-can have normal levels, but inactive HGPRT, or unstable (higher activity in young RBC than old)
what are symptoms of Lesch-Nyhan syndrome?
hyperuricemia, gout, urinary tract stones, and neurological symptoms (mental retardation, spasticity, self-mutilation)
-basis for latter is unknown, but brain cells have much higher levels of purine salvage enzymes than other cells
--may normally use salvage pathways to a greater extent
what is treatment of Lesch-Nyhan syndrome?
allopurinol (for same reason as gout)
-doesn't alleviate neurlogic symptoms
what is adenosine deaminase (ADA) deficiency?
deoxyadenosine and adenosine are abundant b/c not degraded to deoxyinosine and inosine
-instead efficiently converted to nucleotides in WBC
-high levels of dATP inhibit ribonucleotide reductase, which inhibits DNA synthesis, so WBC can't proliferate
-associated with severe combined immunodeficiency (SCID)