deoxyribonucleotidos sintesis y degradacion

7/31/2019 Deoxyribonucleotidos Sintesis y Degradacion

1/19

12. Deoxyribonucleotide biosynthesis andnucleotide catabolism


2/19

dTTP

ADP

GDPCDP

UDP

H2O

NADP+ NADPH

ribonucleotidereductase

X

SH

SH

X

S

S

Ribonucleotide reductase reduces ADP, GDP,CDP & UDP to the deoxyribonucleotides

DNA synthesis also

requires thymine, which

is made from dUDP.

Regeneration of the reduced

enzyme requires several steps

Two cysteines in

the enzyme serveas the reductant.

OHOH

OP -O- P -O-CH2

N

OH

P -O- P -O-CH2O

N

dADP

dGDP

dCDP

dUDP


3/19

QuickTime an d a

TIFF (Uncompressed) decompressorare needed to see this p icture.

1av8.pdb

R2dimer

regulatorysites

R2 R2

O O

R1 R1

The enzyme has two types

of subunits, R1 and R2.

Each of the catalytic sites in ribonucleotide reductasehas a binuclear Fe center and a stable tyrosyl radical

O CHCH2

C=O

NH

O OC

Fe Fe

C

O O CO O

NN

NN

CO O

tyrosylradical

two Fe

atoms with His, Glu & Asp ligands

The catalyticsites are atthe R1-R2interfaces


4/19

Ribonucleotide reductase generates free-radical intermediates

NDP

P -O- P -O-CH2 N

OHOH

O

HOHOH

OP -O- P -O-CH2 N

H H

XH

SH

SH

OH

P -O- P -O-CH2 N

OH

+

XH

S

S

HOH

O

P -O- P -O-CH2 N

H H

X

SH

SH

X

S

S

dNDP

1

2

3

The tyrosine radical

generates another radical(X ) close to the substrate.

Most electron-transfer reactions

of NADH involve hydride ion

transfer.

P -O- P -O-CH2 N

HOH

O

H

Steps 1 & 3: H atom

(H ) transfer

Step 2: hydride ion (H )

transfer or two 1-ereactions

OH-

H+


5/19

Two sets of redox proteins carry electrons fromNADPH to ribonucleotide reductase (RNR)

glutathione

glutathionereductase

glutaredoxinreductase

X

SH

SH

X

S

S

NDP dNDP

S

S

SH

SH

GSSG2 GSH

NADPHNADP

+

glutaredoxin

CO2

H3N-CH-CH2CH2-CO-NH-CH-CO-NH-CH2-CO2

CH2SH+

-

-

Glutathione (g-Glu-Cys-Gly) is

the main redox buffer in the

cytosol of most eukaryotic cells

NADP+NADPH

thioredoxinreductase

S

S

SH

SHthioredoxin

X

SH

SH

X

S

S

RNR

RNR


6/19

Regulation of ribonucleotide reductase at two allosteric sitesbalances the rates of formation of the different deoxyribonucleotides

dTTP inhibits

reduction of CDP &

UDP, and activates

reduction of GDP.

ATP activates

reduction of CDP &

UDP.

dATP inhibits and

ATP activates the

enzyme with all

substrates.

allostericeffectors

substrates

R2 R2

R1 R1

activityregulation

site

substrate-specificitysite

catalyticsite

ATP, dATP,dGTP, dTTP

ADP, CDP,UDP, GDP

SHSH

HSHS

ATP, dATP

-+


7/19

At the activity-regulation site, dATP inhibits RNR and ATPincreases Vmaxfor the reactions of all the ribonucleotides

R1

substrate-specificitysite

R2

catalyticsite

SHSH

activityregulation

site

ADP ATP

dADP dATP

-

+


8/19

At the specificity site, ATP, dATP, dTTP and dGTP tunethe relative affinities of RNR for CDP, UDP, GDP and ADP

+

-

-

-

-

ATP ADP dADP dATP

GDP dGDP dGTP

UDP dUDP dTTP

CDP dCDP dCTP

+

+

+

+


9/19

H2O

PPi

dUMP

dTMP

thymidylatesynthase

N

CHC

O

HN

CHCO

O

OHOH

-O-P-O-CH2

O-

O

N5,N10-methylene-THF

N

CC

O

HN

CHCO

CH3

dTTPATP

dCTPdeaminase

H2O

NH3


nucleosidediphosphate

kinase

C

NH2

N

NCHC

O

CH

CDP dCDP dCTP

ATP ADP

UDP dUDP dUTP

ATP ADP

Thymidylate (dTMP) can be synthesized from either CDP or UDP

Hydrolysis of dUTP looks

wasteful. Why dont cells use

dUTP to make dTTP directly?


10/19

In the process of methylating dUMP, thymidylate synthaseoxidizes N5,N10-methylenetetrahydrofolate to dihydrofolate

dTMP

7,8-dihydrofolateN5,N10-methylene-tetrahydrofolate

dUMP

N

CHC

O

HN

CHCO

OH

-O-P-O-CH2

O-

OO

OH

-O-P-O-CH2

O-

OO

CH3

N

CC

O

HN

CHCO

H

N

H

CH

N

HN

OH

H2N N

N

O

C-NH-(Glu)n

H

H

O

C-NH-(Glu)nN

N

HN

OH

H2N N

N

H

H

H


11/19

Dihydrofolate reductase regenerates tetrahydrofolate,using NADPH as the reductant

N5,N10-methylene-tetrahydrofolate

Serine

Glycine

7,8-dihydrofolate tetrahydrofolateNADPH + H+ NADP+

dihydrofolatereductase

serine hydroxymethyltransferase (PLP)

N

HNN

N

OH

H2N

HN R

H

H

OH

H2N N

N

H

H

NH

N

HN

C

H

H

H

HNHN

HNH2N

OH

N

N

H

H


12/19

Enzymes of nucleotidebiosynthesis provide targets

for cancer chemotherapy

methotrexate

Analog of DHF. Inhibits

dihydrofolate reductase

CO2-

+H3N C H

CH2

O

N=N=CH-C-O-C=O

O

- +

azaserine(O-diazoacetyl-L-serine)

Analog of Gln. Inhibits glutamine

amidotransferases (steps 1 & 4 of

purine biosynthesis, CTP synthase, &

carbamoylphosphate synthetase II).

N

C-FC

O

HN

CHC

O

OH

-O-P-O-CH2O

-

OON

C-FC

O

HNCHC

OH

5-fluorouracil FdUMP

Analog of dUMP. Inhibits

thymidylate synthase.

(in vivo)

H2N-C=O

CO2-

+H3N C H

CH2

CH2

Gln

NCH3

N

HN

NH2

H2N N

NO

C-NH-Glu


13/19

Primates oxidize purines to uric acid for excretion

uric acid

adenosine guanosine

Birds, reptiles and insects also excrete uric acid.

Fish and most terrestrial mammals oxidize uric

acid further before excretion.

NH2

C

CN

N

N

CH

C

NHC

ribose

C

CHN

N

N

CH

C

O

NH2N-C

ribose

O

C

CHN

N

C OH

C

NC

HO NH

CCOH

C

NN

C OH

NC

HO NH

uric acid has several

tautomeric forms

C

CHN

C O

CO

NCO N

H

NH


14/19

Uric acid production from purines

C

CHN

N

N

CH

CO

NH2N-C

ribose

C

CHN

N

C OH

C

O

NC

HO NH

uric acidguanosine

adenosine inosine hypoxanthine

NH2

C

CN

N

N

CH

C

NHC

ribose ribose

C

CHN

N

N

CH

C

O

NHCadenosine

deaminase

C

CHN

N

N

CH

C

O

NHC

H

H2O NH4+

Pi ribose-1-P

C

CHN

N

N

CH

CO

NC

HHO

xanthine

O2 + H2O

O2 + H2O

H2O2

H2O2

xanthineoxidase

xanthineoxidase

Xanthine oxidase contains FAD, a

Mo complex, and two Fe-S centers.

Its substrates are the free purines,

not the nucleosides or nucleotides.


15/19

In the absence of adenosine deaminase, dATP accumulates to high levels.

dATP inhibits ribonucleotide reductase, which prevents synthesis of other

deoxyribonucleotides and cuts off DNA synthesis. Lymphocytes are

particularly susceptible to this inhibition. Untreated SCID is fatal. SCID

was the first human disorder to be addressed by gene therapy.*

Mutations in adenosine deaminase causeSevere Combined Immunodeficiency Disease

deoxy-

adenosine

NH2

C

CN

N

N

CH

C

NHC

deoxyribose deoxyribose

C

CHN

N

N

CH

C

O

NHCadenosine

deaminase

H2O NH4+

uricacid

dATP

ribonucleotides deoxyribonucleotides


DNA

- *see Lehninger, Box 9-2 (p. 336)


16/19

Deposition of sodium uratecrystals in tissues causes gout

allopurinol

C

CHN

N

C

N

C

O

NHC

H

H

Impaired excretion of uric acid results in high levels of uric acid in body fluids. Crystals

of sodium urate form in the toes, kidney and other tissues, causing painful inflamation.

uric acidhypoxanthine xanthine

C

CHN N

C OH

C

O

NC

HO NH

C

CHN

N

N

CH

C

O

NHC

H

C

CHN

N

N

CH

C

O

NC

HHO

O2 + H2O H2O2

xanthine

oxidase

xanthine

oxidase

O2 + H2O H2O2

sodium urate

C

CHN

N

C O-

CO

NC

HO NH

Na+Gout can be treated with

allopurinol, an analog of

hypoxanthine that inhibits

xanthine oxidase.


17/19

Salvage pathways regenerate nucleotides

from free purine and pyrimidine bases

A similar enzyme (hypoxanthine-guanine

phosphoribosyl transferase) works on

hypoxanthine and guanine. Mutations in this

enzyme lead to Lesch-Nyhan syndrome.

adenineNH2

C

CN

N

N

CH

C

NHC

H

adeninephosphoribosyltransferase

PPi

O

O- O-

O

O P O P O-

OHOH

OP O-CH2

5-phosphoribosyl-1-pyrophosphate (PRPP)

NH2

C

CN

N

N

CH

C

NHC

OHOH

OP O-CH2

AMP


18/19

Lesch-Nyhan syndrome

Lesch-Nhyan syndrome, which results from a mutation in the gene forhypoxanthine-guanine ribosylphosphotransferase, is characterized bysevere neurological defects.

mental retardation

self-mutilation

cerebral palsy

elevated uric acid (gout)


19/19

Lesch-Nyhan Syndrome can be diagnosed prenatally

Fetal fibroblasts obtained by amniocentesis are

cultured in the presence of3H-hypoxanthine.

Cells from normal individuals converthypoxanthine into IMP, which procedes to AMP

and GMP and is incorporated into DNA.

Blocked inLesch-Nyhan

syndrome

normal

Lesch-Nyhanhypoxanthine

C

CHN

N

N

CH

C

O

NHC

HIMP

OHOH

OP O-CH2

C

CHN

N

N

CH

C

O

NHC

PRPP PPi

AMP

GMP

DNA

deoxyribonucleotidos sintesis y degradacion

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