ch12-nucleotide_metab_1

8/9/2019 CH12-Nucleotide_metab_1

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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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ch12-nucleotide_metab_1

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