06 May 2008 Nucleic Acid Metabolism Andy Howard Introductory Biochemistry 6 May 2008.

06 May 2008Nucleic Acid Metabolism

Nucleic Acid Metabolism

Andy HowardIntroductory Biochemistry

6 May 2008

06 May 2008Nucleic Acid Metabolism p.2 of 56

What we’ll discuss Pyrimidine synthesis

PRPP Pathway to UMP Regulation Pathway to CTP

Purine synthesis IMP AMP, XMP, GMP Regulation

Reduction of riboNucs to deoxyNucs

dUMP to dTMP Salvage pathways Pyrimidine

catabolism Purine catabolism


PRPP synthetase

Activation of ribose-5-P (see Calvin cycle, etc.) by ATP:-ribose-5-P + ATP PRPP + AMP

Has roles in other systems too

Phosphoribosyl pyrophosphate

PRPP synthetasePDB 2H06

215 kDa hexamerdimer shown; human


Pyrimidine synthesis:carbamoyl aspartate

Uridine is based on orotate,which is derivated fromcarbamoyl aspartate

We’ve already seen the carbamoyl phosphate synthesis back in chapter 17 via carbamoyl phosphate synthetase

Carbamoyl phosphate + aspartate carbamoyl aspartate + Pi

via aspartate transcarbamoylase

Carbamoyl aspartate

Carbamoyl phosphate


Aspartate transcarbamoylase

ATCase is the classic allosteric enzyme

E.coli version is inhibited by pyrimidine nucleotides and activated by ATP

CTP by itself is 50% inhibitory; CTP+ UTP is almost totally inhibitory

ATCasePDB 1D09Trimer of heterotetramers1 heterotetramer shown (cf. fig.18.11)E.coli


Carbamoyl aspartate to dihydroorotate

Carbamoyl aspartate dehydrates and cyclizes to L-dihydroorotate via dihydroorotase

TIM barrel protein

Dihydro-orotate

PDB 1XGE76 kDa dimerE.coli


Dihydroorotate to orotate

Ubiquinone acts as oxidizing agent reducing the 5 & 6 Carbons via dihydroorotate dehydrogenase

Some versions incorporate FMN

PDB 2E6F69 kDa dimerTrypanosoma cruzi


Adding phosphoribose

Orotate + PRPP orotidine 5’-monophosphate + PPi

Usual argument re pyrophosphate hydrolysis

Enzyme: orotidine phosphoribosyl transferase Orotidine 5’-

monophosphate

PDB 2PS150 kDa dimerYeast


Decarboxylation OMP

decarboxylated to form UMP via OMP decarboxylase

Bacterial forms are TIM barrel proteins

Acceleration is 1017-fold relative to uncatalyzed rate

PDB 1KLY54 kDa dimerMethanobacteriumthermoautotrophicum


Eukaryotic variation Orotate produced in the

mitochondrion moves to the cytosol

UMP synthase combines the last two reactions—orotidine to OMP to UMP

OMP decarboxylasedomainof UMP synthasePDB 2P1F64 kDa dimerhuman


UMP to UTP Uridylate kinase converts

UMP to UDP:UMP + ATP UDP + ADPenzyme is related to several amino acid kinases

Nucleoside diphosphate kinase exchanges di for tri:UDP + ATP UTP +ADP(non-specific enzyme)

Uridylate kinasePDB 2A1F163 kDa hexamerHaemophilusinfluenzae


CTP synthetase

UTP + gln + ATP CTP + glu + ADP + Pi

Glutamine side-chain is amine donor

ATP provides energy sandwich (Rossmann) Enzyme is inhibited by CTP In E.coli, it’s activated by

GTP (makes sense!)

PDB 1S1M240 kDa tetramerdimer shownE.coli


Purine synthesis Considerably more complex than

pyrimidine synthesis More atoms to condense and two rings to

make More ATP to sacrifice during synthesis Several synthetase (ligase) reactions

require ATP Based on PRPP, gln, 10-formyl THF, asp


PRPP + gln to phospho-ribosylamine

PRPP aminated:PRPP + gln glu + PPi +5-phospho--D-ribosylaminevia glutamine-PRPP amidotransferase

transferase structure Product is unstable

(lasts seconds!)

1

PDB 1ECF120 kDa tetramerdimer shownE.coli


Phospho-ribosylamine to GAR

Amine condenses with glycine to form glycinamide ribonucleotide (GAR)

ATP hydrolysis drives GAR synthetase reaction to the right PDB 2YRX

50 kDa monomerGeobacillus kaustophilus

2


Formylation of GAR

10-formyl THF donates a formyl (-CH=O) group to end nitrogen with the help of GAR transformylase to form formylglycinamide ribonucleotide (FGAR)

Rossmann

FGAR

PDB 1MEO47 kDa dimer

human

3


FGAR to FGAM

Glutamine sidechain is source of N for C=O exchanging to C=NH via FGAM synthetase to form formylglycinamidine ribonucleotide (FGAM):FGAR + gln + ATP + H2O FGAM + glu + ADP + Pi

PurS component ofFGAM synthetasePDB 1GTD37.4 kDa tetramerdimer shown

Methanobacterium

FGAM

4


FGAM to AIR Cyclize FGAM to

aminoimidazole ribonucleotide

ATP drives the AIR synthetase reaction:

FGAM + ATP AIR + H2O + ADP + Pi

E.C. in Wikipedia is wrong:it should be 6.3.3.1

PDB 2V9Y147 kDa tetramerdimer shownhuman

Aminoimidazoleribonucleotide

5


AIR to CAIR AIR is carboxylated;

expenditure of an ATP:AIR + HCO3

- + ATP carboxyaminoimidazole ribonucleotide + ADP + Pi + 2H+

AIR carboxylase E.coli version is two

enzymes; eukaryotes have a single enzyme

No cofactors!

PDB 2NSH149 kDa octamermonomer shownE.coli

CAIR

6


CAIR+asp to SAICAR

CAIR + asp + ATP aminoimidazole succinylocarboxamide ribonucleotide + ADP + Pi

Enzyme is SAICAR synthetase

Domain 1: homolog ofphosphorylasekinase

Domain 2: ATP-bindingPDB 2CNQ34 kDamonomeryeast

7


SAICAR to AICAR SAICAR aminoimidazole

carboxamide ribonucleotide + fumarate

Enzyme is adenylosuccinate lyase

Net result of two reactions is just replacing acid with amide;

That’s like first 2 reactions in urea cycle, except ADP, not AMP, is the product PDB 2PTR

203 kDa tetramerdimer shown; E.coli

8


AICAR to FAICAR 10-formylTHF donates HC=O:

AICAR + 10-formylTHF formamidoimidazole carboxamide ribonucleotide + THF

Enzyme: AICAR transformylase Like step 3 Generally a bifunctional enzyme

combined with next step This part is like cytidine

deaminase (see below)

9

PDB 1THZ130 kDa dimer

chicken


FAICAR to IMP We made it:

FAICAR inosine 5’-monosphosphate + H2O

Bifunctional enzyme; this part is called IMP cyclohydrolase or inosicase

Hydrolase part is like methylglyoxal synthase PDB 1PL0

260 kDa tetramerdimer shown; human

10


So now we have a purine. What next?

Enzymatic conversions to AMP or GMP;Details on next few slides

AMP and GMP can be further phosphorylated to make ADP, GDP with specific kinases (adenylate kinase and guanylate kinase)

GTP made with broad-spectrum nucleoside diphosphate kinase


IMP to adenylosuccinate

IMP + aspartate + GTP adenylosuccinate + GDP + Pi

Enzyme is adenylosuccinate synthase

Similar to step 7 in IMP synthesis

PDB 2V40101 kDa dimermonomer shownhuman


Adenylosuccinate to AMP

Adenylosuccinate AMP + fumarate

Like reaction 8 in the IMP pathway; in fact, it uses the same enzyme, adenylosuccinate lyase

PDB 2PTR203 kDa tetramerdimer shown; E.coli


IMP to XMP

IMP + H2O + NAD+ Xanthosine monophosphate + NADH + H+

Enzyme: IMP dehydrogenase

TIM-barrel, aldolase-like protein

PDB 1ME8221 kDa tetramer;monomer shownTritrichomonas foetus


XMP to GMP

XMP + gln + H2O + ATP GMP + glu + AMP + PPi

Enzyme: GMP synthetase

Typical 3-layer sandwich

PDB 2DPL68 kDa dimerPyrococcus horikoshii


Adenylate kinase

Reminder:ATP + AMP 2 ADP

Metal ions play a role in enzyme structure

Enzymes like this need to shield their active sites from water to avoid pointless hydrolysis of ATP

PDB 1ZIN24 kDa monomerBacillus stearothermophilus


Guanylate kinase GMP + ATP GDP + ADP “P-loop”-containing ATP-

binding proteins Rossmann fold

PDB 2QOR22 kDa monomerPlasmodium vivax


Purine control I: IMP level

Note that GTP is a cosubstrate in making AMP from IMP

ATP is a cosubstrate in making GMP from IMP

So this helps balance the 2 products


Purine control II:feedback inhibition

PRPP synthetase inhibited by purines, but only at unrealistic concentrations of [Pur]

Step 1 (gln-PRPP amidotransferase) is allosterically inhibited by IMP, AMP, GMP

Adenylosuccinate synthetase is inhibited by AMP

XMP and GMP inhibit IMP dehydrogenase


Making deoxyribonucleotides

Conversions of nucleotides to deoxynucleotides occurs at the diphosphate level

Reichard showed that most organisms have a single ribonucleotide reductase that converts ADP, GDP, CDP, UDP to dADP, dGDP, dCDP, and dUDP

NADPH is the reducing agent


Ribonucleotide reductase heterotetramer

2 RNR1 subunits; each has a helical 220-aa domain 10-strand 480-aa structure

(thiols here) 5-strand 70-aa structure

2 RNR2 subunits; each has A diferric ion center A stable tyrosyl free radical

RNR1PDB 1R1R

258 kDa dimerE.coli

RNR2PDB 1PJ0

82 kDa dimerE.coli


Mechanism of RNR (box 18.3)

Y122 in RNR2 is converted to stable free radical

Radical transmitted to RNR1 cys439 Cys439 reacts with substrate 3’-OH to form

free radical at C3’ Substrate dehydrates to carbonyl at C3’ and

free radical at C2’; S- formed at Cys462 Disulfide formed between Cys462,Cys225;

radical regenerated at Cys439


Ribonucleotide reductase: control

ATP, dATP, dTTP, and dGTP act as allosteric modulators by binding to two regulatory sites on the enzyme

Activity site (A) regulates activity of catalytic site When ATP binds at A, activity goes up When dATP binds at A, activity inhibited overall

Specificity site (S) controls which substrates can be turned over ATP at A + ATP or dATP at S : pyrimidines only dTTP at S : activates reduction of GDP dGTP at S : activates reduction of ADP


dUDP to dUMP(for making dTMP)

dTMP formed at monophosphate level(from dUMP)

dUMP derived three ways: dUDP + ADP dUMP + ATP dUDP + ATP dUTP + ADP

dUTP + H2O dUMP + PPi

dCMP + H2O dUMP + NH4+


Thymidylate synthase reaction (fig.18.15)

dUMP + 5,10-methyleneTHF dTMP + 7,8-dihydrofolate

Unusual THF reaction in that cofactor gets oxidized as well as giving up a carbon CH2 from 5,10-methylene group extra H from C6

So DHF must be reduced back to THF via DHFR and get its methylene back from SHMT

5,10-methylene THF

dihydrofolate


Thymidylate synthase Generally the controlling step in

DNA synthesis because [dTTP] < other [deoxynucleoside triphosphates]

Therefore a target for cancer chemotherapy and other therapies that target rapidly-dividing cells

Enzyme is a 2-layer sandwich

PDB 2G8O58 kDa dimer

E.coli(with dUMP

and cofactor analog)


Thymidylate synthase and drug design

Both folate analogs and dUMP analogs can interfere with (DHFR SHMT dTMP synthase … ) cycle

5-fluorouracil is specific to thymidylate synthase


DHFR Converts DHF to THF:

DHF + NADPH + H+ <->THF + NADP+

SHMT then converts THF to 5,10-methyleneTHF

3-layer sandwich Often the target for drug design

as well Eukaryotic DHFR also catalyzes

folate DHF Prokaryotic DHFR doesn’t;

DHF derived by another mechanism in bacteria

PDB 1KMV20 kDa monomerhuman

folate


Special case:protozoan

TSynth/DHFR Bifunctional enzyme:

Thymidylate synthase Dihydrofolate reductase

Presumably some entropic advantage

Maybe electrostatics too, allowing the negative charges on DHF to tunnel through;but cf. Atreya et al (2003) J.Biol.Chem. 278:28901.

DHFR-TSPDB 1J3K

104 kDa dimer

Plasmodium falciparum


Recovery pathway to dTMP

Deoxythymidine can be phosphorylated by thymidine kinase:deoxythymidine + ATP dTMP + ADP

Labeled thymidine is convenient for monitoring intracellular synthesis of DNA because thymidine enters cells easily

PDB 1E2K73 kDa monomerHerpes simplex virus


Fates of polynucleotides

Nucleic acids hydrolyzed to mononucleotides via nucleases

Mononucleotides are dephosphorylated via nucleotidases and phosphatases

Resulting nucleosides are deglycosylated via nucleosideases or nucleoside phosphorylases

Resulting bases are sent either into salvage pathways or get degraded and excreted


Salvage pathways We can describe them, and we will: but

why do they matter so much? They provide energy savings relative to de novo

synthesis (think of all the ATP we used in making IMP!)

Considerable medical significance to interference with these pathways

Intracellular nucleic acid bases are usually recycled; dietary bases are usually broken down and excess nitrogen excreted


Orotate phosphoribosyl transferase

Principal salvage enzyme for pyrimidines

Orotate + PRPP -> OMP + PPi

OMP can then reenter UMP synthetic pathway (decarboxylation to UMP, then form UDP and CDP)

Same enzyme can aact on other pyrimidines to make nucleotides:Pyr + PRPP -> PyrMP + PPi

PDB 2 PS150 kDa dimerYeast


Pyrimidine interconversions (fig. 18.19) All phosphorylations & dephosphorylations can

and do happen UTP can be aminated to CTP CDP and UDP can be reduced to dCDP and

dUDP dCMP can deaminate to dUMP Cytidine can be converted to uridine dUMP can be methylated to dTMP


Purine nucleotide salvage

Two phosphoribosyl transferases convert adenine, guanine, and hypoxanthine to AMP, GMP, and IMP

Adenine phosphoribosyl transferase is specific

HGPRT accepts both hypoxanthine and guanine

Hypoxanthine-guaninephosphoribosyltransferasePDB 1FSG102 kDa tetramerdimer shownToxoplasma gondii


Purine Interconnections(fig. 18.18)

All phosphorylations and dephosphorylations can and do occur

ADP and GDP can be reduced to dADP and dGDP

AMP can deaminated to IMP (new) IMP can be aminated to AMP IMP can oxidized to XMP XMP can be aminated to GMP Guanine, adenine can be

phosphoribosylated to GMP and AMP


Fates of CMP and cytidine

CMP’s phosphate can be hydrolyzed off

That’s followed by deamination of cytidine to make uridine Catalyzed by cytidine

deaminase Another sandwich

protein

Cytidine deaminasePDB 2FR564 kDa tetramerMouse


Hydrolysis of U, dU and dT

Glycosidic bond in uridine or thymidine is hydrolyzed by phosphate: Uridine + Pi -> -D-ribose-1-P +

uracil Enyzme is uridine

phosphorylase Similar enzyme handles

deoxyuridine Similar reaction using

thymidine phosphorylase yields thymine + -D-deoxyribose-1-P

Uridine phosphorylasePDB 1RXY167 kDa hexamerDimer shownE.coli


Uracil to acetyl CoA;thymine to succinyl CoA Reduced to dihydrouracil and

dihydrothymine Hydrated and ring-opened to

ureidopropionate or ureidoisobutyrate

Eliminate bicarbonate and ammonium to yield -alanine or -aminoisobutyrate

Several reactions from there to acetyl CoA and succinyl CoA

Dihydro-pyrimidinasePDB 1GKP302 kDa hexamerThermus


Purine catabolism

Nucleoside or deoxynucleoside + phosphate base + (D)-ribose 1-P

Hypoxanthine and guanine both lead to uric acid as a product

Uric acid is the final excreted nitrogenous compound in primates and birds and some reptiles

Other organisms catabolize it further

Uric acid


Uric acid to allantoin

Urate oxidase:urate + 2H2O + O2 allantoin + H2O2 + CO2

That’s the final product in a lot of mammals, turtles, some insects, gastropods

Other organisms catabolize allantoin further; we’ll talk about that on Thursday

Uric acid

Allantoin

Urate oxidase134 kDa tetramermonomer shown

Aspergillus flavus


Lesch-Nyhan syndrome

Complete lack of hypoxanthine-guanine phosphoribosyl transferase

So hypoxanthine and guanine are degraded to uric acid rather than being built back up into IMP and GMP

Leads to dangerous buildup of uric acid in nervous tissue

Neurological effects are severe and poorly understood

Michael Lesch

William Nyhan


Gout

Accumulation of sodium urate and uric acid, both of which are only moderately soluble

Arises from inadequate (~10%) functionality of HGPRT, so that urate accumulates in peripheral tissues, particularly the feet

Sodium urate crystalsaccumulating

Sodium urate

Benjamin Franklin(celebrated gout sufferer)

06 May 2008 Nucleic Acid Metabolism Andy Howard Introductory Biochemistry 6 May 2008.

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