12.

Deoxyribonucleotide biosynthesis and nucleotide catabolism

Ribonucleotide reductase reduces ADP, GDP, CDP & UDP to the deoxyribonucleotides
P -O- P -O-CH2

N
O OH

P -O- P -O-CH2

N
O

OH

OH

ADP GDP CDP UDP Two cysteines in the enzyme serve as the reductant. X ribonucleotide SH reductase SH

dADP

H2O

dGDP dCDP

X S S

dUDP

dTTP

NADP+

NADPH

Regeneration of the reduced enzyme requires several steps

DNA synthesis also requires thymine, which is made from dUDP.

Each of the catalytic sites in ribonucleotide reductase has a binuclear Fe center and a stable tyrosyl radical
The enzyme has two types of subunits, R1 and R2. regulatory sites R1
O

R2 dimer

R1 The catalytic sites are at the R1-R2 interfaces

R2

R2

QuickTime an d a TIFF (Uncompressed) decompressor are need ed to see this p icture .

NH CHCH2 C=O O

O N

O Fe

O Fe O C

O N

O
1av8.pdb

tyrosyl radical

two Fe atoms with His, Glu & Asp ligands

Ribonucleotide reductase generates free-radical intermediates

P -O- P -O-CH2

N
O

P -O- P -O-CH2

N
O

NDP

H
OH

H
OH

X SH SH

OH

H
OH

The tyrosine radical generates another radical (X) close to the substrate.

XH SH SH

OH P -O- P -O-CH2

N
O

X S S

OH

P -O- P -O-CH2

N
O
3

XH S S

2 H+

dNDP

H
OH H

H
P -O- P -O-CH2

N
O

Steps 1 & 3: H atom (H) transfer Step 2: hydride ion (H ) transfer or two 1-e reactions

Most electron-transfer reactions of NADH involve hydride ion transfer.

OH
H

Two sets of redox proteins carry electrons from NADPH to ribonucleotide reductase (RNR)
NDP X SH dNDP X S
RNR

X SH SH

X S S

RNR

SH

S
thioredoxin

S S

SH SH

glutaredoxin

S
S

SH
SH
thioredoxin reductase

glutaredoxin reductase glutathione

NADPH

NADP+

2 GSH

GSSG

glutathione reductase

CH2SH + H3N-CH-CH2CH2-CO-NH-CH-CO-NH-CH2-CO2 CO 2

NADP+

NADPH

Glutathione (g-Glu-Cys-Gly) is the main redox buffer in the cytosol of most eukaryotic cells

Regulation of ribonucleotide reductase at two allosteric sites balances the rates of formation of the different deoxyribonucleotides
allosteric effectors

substratespecificity site
activity regulation site catalytic site HS HS

R1

R1

ATP, dATP, dGTP, dTTP

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.

ATP, dATP

+
SH SH

ADP, CDP, UDP, GDP substrates

R2

R2

At the activity-regulation site, dATP inhibits RNR and ATP increases Vmax for the reactions of all the ribonucleotides

substratespecificity site R1 activity regulation site SH SH catalytic site R2

+
ADP dADP ATP dATP

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

+
CDP

+
-

dCDP

dCTP

+
UDP

dUDP

dTTP

GDP

+
dGDP dGTP

+
ATP ADP dADP dATP

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

NH2 C N O C N CH CH

ATP ADP CDP

ribonucleotide reductase

dCDP
nucleoside diphosphate kinase

dCTP

H2O dCTP deaminase

NH3 O C
HN CH C CH OO N -O-P-O-CH 2 O O OH OH HN O C C N CH3

UDP

dUDP ATP ADP

dUTP
H2O PPi

dUMP
N5,N10-methyleneTHF thymidylate synthase

Hydrolysis of dUTP looks wasteful. Why dont cells use dUTP to make dTTP directly?

CH

dTMP

ATP

dTTP

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

H N N OH N

H2N
N

H H H
O

H2N N
C-NH-(Glu)n

H N
N

H H
O

HC H N5,N10-methylenetetrahydrofolate

OH

HN

C-NH-(Glu)n

7,8-dihydrofolate O C HN CH HN O C C CH3

C CH OO N -O-P-O-CH 2 O O dUMP OH

C CH OO N -O-P-O-CH 2 O O dTMP OH

Dihydrofolate reductase regenerates tetrahydrofolate, using NADPH as the reductant

H2N N OH 7,8-dihydrofolate N H N N HN R NADPH + H+ NADP+ H H dihydrofolate reductase H2N N OH N H N N H H H H HN

tetrahydrofolate Serine

serine hydroxymethyl transferase (PLP) Glycine

H2N
N5,N10-methylenetetrahydrofolate N

H N N

H H H N

OH

HC H

Enzymes of nucleotide biosynthesis provide targets for cancer chemotherapy

H2N

H N methotrexate N O C-NH-Glu

N
NH2 O

CH3 N

O C HN O C N H C-F CH (in vivo)

C HN C-F

Analog of DHF. Inhibits dihydrofolate reductase

C CH OO N -O-P-O-CH 2 O Analog of dUMP. Inhibits O

5-fluorouracil

FdUMP CO2-

thymidylate synthase.
OH CO2-

+H N C H azaserine 3 (O-diazoacetylCH2 L-serine) O - + N=N=CH-C-O-C=O

Analog of Gln. Inhibits glutamine amidotransferases (steps 1 & 4 of purine biosynthesis, CTP synthase, & carbamoylphosphate synthetase II).

+H N 3

C H
CH2 CH2

H2N-C=O Gln

Primates oxidize purines to uric acid for excretion

NH2 C N HC N C C N CH N HN H2N-C

O
C C N C N CH N HN HO uric acid C

O C C N C N C OH N H

ribose
adenosine

ribose
guanosine

OH C

C N O C C

N
C OH N H H N C O

Birds, reptiles and insects also excrete uric acid. Fish and most terrestrial mammals oxidize uric acid further before excretion.
uric acid has several tautomeric forms

HO

HN

C O C N N H

Uric acid production from purines

adenosine NH2 C N C N C N CH N ribose adenosine deaminase HN HC N H2O NH4+ inosine O hypoxanthine Pi CH N ribose-1-P HN HC N O C C C N CH N H

C
C C

HC

ribose
O O2 + H2O H2O2 xanthine oxidase

O
C HN H2N-C C N C N CH N

guanosine

ribose

N C HN CH C C HO N N H xanthine O2 + H2O

O
C HN HO C N

uric acid C C N C OH N H

Xanthine oxidase contains FAD, a Mo complex, and two Fe-S centers. Its substrates are the free purines, not the nucleosides or nucleotides.

xanthine H2O2 oxidase

Mutations in adenosine deaminase cause Severe Combined Immunodeficiency Disease

deoxyadenosine N HC N NH2 C C C N H2O NH4+ HN HC N

O
C C C N CH N deoxyribose uric acid

CH
N

adenosine deaminase

deoxyribose

In the absence of adenosine deaminase, dATP accumulates to high levels. dATP inhibits ribonucleotide reductase, which prevents synthesis of other
dATP

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.*

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

ribonucleotide reductase ribonucleotides deoxyribonucleotides

DNA

Deposition of sodium urate crystals in tissues causes gout

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.
hypoxanthine xanthine O C N C N N H xanthine oxidase C uric acid

O C
HN HC

CH

C C HN HN CH C OH C C C C HO HO N N N N H H O2 + H2O H2O2 O2 + H2O H2O2

xanthine oxidase

O C N

O C HN HC N C C H C N

N H allopurinol

Gout can be treated with allopurinol, an analog of hypoxanthine that inhibits xanthine oxidase.

C HN HO C C

N H sodium urate

Na+ C O-

Salvage pathways regenerate nucleotides from free purine and pyrimidine bases
NH2 adenine N C C N C N CH N H OP O-CH2 O OH OPPi OH AMP P adenine phosphoribosyl transferase

NH2
C N HC C N CH N

HC

N
O-CH2

OPOPOO O OH

OH

5-phosphoribosyl-1pyrophosphate (PRPP)

A similar enzyme (hypoxanthine-guanine phosphoribosyl transferase) works on hypoxanthine and guanine. Mutations in this enzyme lead to Lesch-Nyhan syndrome.

Lesch-Nyhan syndrome

Lesch-Nhyan syndrome, which results from a mutation in the gene for hypoxanthine-guanine ribosylphosphotransferase, is characterized by severe neurological defects.

mental retardation
self-mutilation cerebral palsy elevated uric acid (gout)

Lesch-Nyhan Syndrome can be diagnosed prenatally

Fetal fibroblasts obtained by amniocentesis are cultured in the presence of 3H-hypoxanthine. Cells from normal individuals convert hypoxanthine into IMP, which procedes to AMP and GMP and is incorporated into DNA.
O C HN HC N C C N CH P PRPP PPi HN HC O-CH2 N O C C C O OH N CH

normal

N H hypoxanthine

N
IMP Lesch-Nyhan

Blocked in Lesch-Nyhan syndrome

OH

AMP DNA GMP