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Nucleotide MetabolismNucleotide Metabolism
Chatchawin Petchlert, Ph.D.Chatchawin Petchlert, Ph.D.
Dept. of BiochemistryDept. of Biochemistry
Faculty of Science, Burapha UniversityFaculty of Science, Burapha University
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Biosynthetic routes :Biosynthetic routes :
De novo and Salvage pathwaysDe novo and Salvage pathways
De novo pathwaysDe novo pathwaysAlmost all cell types have the ability to synthesize purine and pyrimidine
nucleotides from low molecular weight precursors in amounts sufficient
for their own needs.
The de novo pathways are almost identical in all organisms.
Salvage pathwaysSalvage pathwaysMost organisms have the ability to synthesize nucleotides from
nucleosides or bases that become available through the diet or fromdegredation of nucleic acids.
In animals, the extracellular hydrolysis of ingested nucleic acids
represents the major route by which bases become available.
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Reutilization and catabolism of purine and pyrimidine basesReutilization and catabolism of purine and pyrimidine bases
blue-catabolismred-salvage pathways
endonucleases:
pancreatic RNAse
pancreatic DNAse
phosphodiesterases:
usually non-specific
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PRPP: a central metabolite in de novo and salvage pathwaysPRPP: a central metabolite in de novo and salvage pathways
Roles of PRPP: his and trp biosynthesis, nucleobase salvage pathways, de
novo synthesis of nucleotides
PRPP synthetase
Enzyme inhibited by AMP, ADP, and GDP. InE. coli, expression is repressed
by PurR repressor bound to either guanine or hypoxanthine.
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Example of a salvage pathway: guanine phosphoribosyl transferaseExample of a salvage pathway: guanine phosphoribosyl transferase
In vivo, the reaction is driven to the right by the action of pyrophosphatase
Shown: HGPRT, cells also have a APRT.
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De novo biosynthesis of purines: low molecular weightDe novo biosynthesis of purines: low molecular weight
precursors of the purine ring atomsprecursors of the purine ring atoms
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Synthesis of IMPSynthesis of IMP
The base in IMP is called
hypoxanthine
Note: purine ring built up at
nucleotide level.
precursors:
glutamine (twice)
glycineN10-formyl-THF (twice)
HCO3aspartate
In vertebrates, 2,3,5 catalyzed
by trifunctional enzyme,6,7 catalyzed by bifunctional
enzyme.
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Proposed reaction mechanism for FGAM synthetaseProposed reaction mechanism for FGAM synthetase
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The transformylation reactions are catalyzed by a multiprotein complexThe transformylation reactions are catalyzed by a multiprotein complex
components of the complex:
GAR transformylase (3)
AICAR transformylase (9)
serine hydroxymethyl transferase, trifunctional formylmethenyl-
methylene-THF synthase (activities shown with asterisk)
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Pathways from IMP to AMP and GMPPathways from IMP to AMP and GMP
G-1: IMP dehydrogenase
G-2: XMP aminaseA-1: adenylosuccinate
synthetase
A-2: adenylosuccinate lyase
Note:Note:GTP used to make AMP,
ATP used to make GMP.
Also, feedback inhibition by
AMP and GMP.
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Pathways from AMP and GMP to ATP and GTPPathways from AMP and GMP to ATP and GTP
Conversion to diphosphate involves specific kinases:
GMP + ATP GDP + ADP Guanylate kinase
AMP + ATP 2 ADP Adenylate kinase
Conversion to triphosphate by Nucleoside diphosphate kinase (NDK):
GDP + ATP GTP + ADP (G0= 0
ping pong reaction mechanism with phospho-his intermediate.
NDK also works with pyrimidine nucleotides and is driven by mass action.
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Allosteric regulation of purine de novo synthesisAllosteric regulation of purine de novo synthesis
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Purine degredationPurine degredation
AMP deamination in muscle, hydrolysis in other tissues.AMP deamination in muscle, hydrolysis in other tissues.
Xanthine oxidase:contains FAD, molybdenum, and nonXanthine oxidase:contains FAD, molybdenum, and non--heme iron.heme iron.
In primates, uric acid is the end product, which is excreted.In primates, uric acid is the end product, which is excreted.
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Purine degredation in other animalsPurine degredation in other animals
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Clinical disorders of purine metabolismClinical disorders of purine metabolism
Excessive accumulation of uric acid:Excessive accumulation of uric acid: GoutGout
The three defects shown each result in elevated de novo purine biosynthesis
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Common treatment forCommon treatment for goutgout: allopurinol: allopurinol
Allopurinol is an analogue of hypoxanthine that strongly inhibitsAllopurinol is an analogue of hypoxanthine that strongly inhibits
xanthine oxidase. Xanthine and hypoxanthine, which are soluble, arexanthine oxidase. Xanthine and hypoxanthine, which are soluble, are
accumulated and excreted.accumulated and excreted.
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Diseases of purine metabolism (continued)Diseases of purine metabolism (continued)
LeschLesch--Nyhan SyndromeNyhan Syndrome: Severe HGPRT deficiency
In addition to symptoms of gout, patients display severe behavioraldisorders, learning disorder, aggressiveness and hostility, including self-
directed. Patients must be restrained to prevent self-mutilation. Reason
for the behavioral disorder is unknown.
X-linked trait (HGPRT gene is on X chromosome).
Severe combined immune deficiency (SCID)Severe combined immune deficiency (SCID): lack of adenosine
deaminase (ADA).
Lack of ADA causes accumulation of deoxyadenosine. Immune cells,
which have potent salvage pathways, accumulate dATP, which blocks
production of other dNTPs by its action on ribonucleotide reductase.
Immune cells cant replicate their DNA, and thus cant mount animmune response.
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De novo pyrimidine biosynthesisDe novo pyrimidine biosynthesis
Pyrimidine ring is assembled as the free base, orotic acid, which is
them converted to the nucleotide orotidine monophosphate (OMP).
The pathway is unbranched. UTP is a substrate for formation of
CTP.
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De novo synthesis of pyrimidinesDe novo synthesis of pyrimidines
1: carbamyl phosphate
synthase
2: aspartate
transcarbamylase
3: dihydroorotase
4: dihydroorotate DH
5: orotatephosphoribosyl
tranferase
6: orotidylate
decarboxylase
7: UMP kinase
8: NDK9: CTP synthetase
CAD=1,2,3
5 +6=single protein
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Reactions catalyzed by eukaryotic dihydroorotate dehydrogenaseReactions catalyzed by eukaryotic dihydroorotate dehydrogenase
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Proposed catalytic mechanism for OMP decarboxylaseProposed catalytic mechanism for OMP decarboxylase
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Regulation of pyrimidine de novo synthesisRegulation of pyrimidine de novo synthesis
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Catabolism of pyrimidinesCatabolism of pyrimidines
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Overview of dNTP biosynthesisOverview of dNTP biosynthesis
One enzyme, ribonucleotide reductase,
reduces all four ribonucleotides to theirdeoxyribo derivitives.
A free radical mechanism is involved
in the ribonucleotide reductase
reaction.
There are three classes of ribonucleotide
reductase enzymes in nature:
Class I: tyrosine radical, uses NDP
Class II: adenosylcobalamin. uses NTPs
(cyanobacteria, some bacteria,Euglena).
Class III: SAM and Fe-S to generate
radical, uses NTPs.
(anaerobes and fac. anaerobes).
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Structure of rNDP reductase (Structure of rNDP reductase (E. coliE. coli, Class I), Class I)
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Proposed mechanism for rNDP reductaseProposed mechanism for rNDP reductase
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Proposed reaction mechanism for ribonucleotide reductaseProposed reaction mechanism for ribonucleotide reductase
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Sources of reducing power for rNDP reductaseSources of reducing power for rNDP reductase
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Biological activities ofBiological activities ofthioredoxinthioredoxin
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Regulation of activities of mammalian rNDP reductaseRegulation of activities of mammalian rNDP reductase
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Salvage and de novo pathways to thymine nucleotidesSalvage and de novo pathways to thymine nucleotides
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Substrate recognition by dUTPaseSubstrate recognition by dUTPase
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Relationship between thymidylate synthaseRelationship between thymidylate synthase
and enzymes of tetrahydrofolate metabolismand enzymes of tetrahydrofolate metabolism
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Catalytic mechanism ofCatalytic mechanism of
thymidylate synthasethymidylate synthase
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Regeneration of N5, N10Regeneration of N5, N10--methylenetetrahydrofolatemethylenetetrahydrofolate
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Biosynthesis ofBiosynthesis of
NADNAD++ and NADPand NADP++
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Biosynthesis of CoABiosynthesis of CoA
from pantothenic acidfrom pantothenic acid
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LeschLesch--Nyhan SyndromeNyhan Syndrome
XX--linked recessive genelinked recessive gene
HGPRT deficiencyHGPRT deficiency pp potentpotent
PR
PPPR
PPoo
, IMP/GMP, IMP/GMPqq
Purine de novo synthesis is activatedPurine de novo synthesis is activated
Uric acidUric acid oo oo oo
Hyperuricemia, gout, urinary bladderHyperuricemia, gout, urinary bladder
stone, neural defectsstone, neural defects
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GoutGout
HyperuricemiaHyperuricemiaPRPP synthetase defectPRPP synthetase defect
HGPRT inactiveHGPRT inactive pp PRPPPRPPoo
Etc.Etc.
ColchicineColchicine
Uricosuric agent (probenecideUricosuric agent (probenecide
)) AllopurinolAllopurinol
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