NucleotidesNucleotides:: Synthesis and Synthesis and DegradationDegradation

Roles of Nucleotides

•Precursors to nucleic acids (genetic material and non-protein enzymes).

•Currency in energy metabolism (eg. ATP, GTP).

•Carriers of activated metabolites for biosynthesis (eg. CDP, UDP).

•Structural moieties of coenzymes (eg. NAD, CoA).

•Metabolic regulators and signal molecules (eg. cAMP, cGMP, ppGpp).

Biosynthetic routes: De novo and salvage pathways

De 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 pathwaysMost organisms have the ability to synthesize nucleotides from nucleosides or bases that become available through the diet or from degredation of nucleic acids.

In animals, the extracellular hydrolysis of ingested nucleic acids represents the major route by which bases become available.

Reutilization and catabolism of purine and pyrimidine bases

blue-catabolismred-salvage pathways

endonucleases:pancreatic RNAsepancreatic DNAse

phosphodiesterases:usually non-specific

PRPP: 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 inhinited by AMP, ADP, and GDP. In E. coli, expression is repressedby PurR repressor bound to either guanine or hypoxanthine.

Purine Nucleotide Synthesis

OH

H

H

CH2

OH OH

H HO

O2-O3P

-D-Ribose-5-Phosphate (R5P)

O

H

H

CH2

OH OH

H HO

O2-O3P

5-Phosphoribosyl--pyrophosphate (PRPP)

P

O

O

O P

O

O

O

ATP

AMP

RibosePhosphatePyrophosphokinase

H

NH2

H

CH2

OH OH

H HO

O2-O3P

-5-Phosphoribosylamine (PRA)

AmidophosphoribosylTransferase

Glutamine + H2O

Glutamate + PPi

H

NH

H

CH2

OH OH

H HO

O2-O3P

CO

H2C NH2

Glycinamide Ribotide (GAR)

GAR Synthetase

Glycine + ATP

ADP+ Pi

H2C

CNH

O

CH

HN

O

Ribose-5-Phosphate

Formylglycinamide ribotide (FGAR)

H2C

CNH

O

CH

HN

HN

Ribose-5-Phosphate

Formylglycinamidine ribotide (FGAM)

THFN10-Formyl-THF

GAR Transformylase

ATP +Glutamine +H2O

ADP +Glutamate + Pi

FGAM Synthetase

HC

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole Ribotide (AIR)

ATP

ADP + Pi

AIR Synthetase

C

CN

CH

N

H2N

OOC

Ribose-5-Phosphate

4

5

Carboxyamidoimidazole Ribotide (CAIR)

ATP+HCO3

ADP + PiAIR Car boxylase

Aspartate+ ATP

ADP+ Pi

SAICAR Synthetase

AdenylosuccinateLyase

Fumarate

C

CN

CH

N

NH

Ribose-5-Phosphate

4

5

5-Formaminoimidazole-4-carboxamideribotide (FAICAR)

CH2N

O

CH

O

C

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole-4-carboxamideribotide (AICAR)

CH2N

O

C

CN

CH

N

H2N

CNH

O

HC

COO

CH2

COO

Ribose-5-Phosphate

4

5

5-Aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR)

THF

AICAR Transformylase

N10-Formyl-

THF

Inosine Monophosphate (IMP)

HN

HCN

C

CC

N

CH

N

O

4

5

HH

CH2

OH OH

H HOO2-O3P

IMPCyclohydrolase

H2O

Example 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.

De novo biosynthesis of purines: low molecular weight precursors of the purine ring atoms

Synthesis of IMP

The base in IMP is called hypoxanthine

Note: purine ring built up atnucleotide level.

precursors: glutamine (twice)glycineN10-formyl-THF (twice)HCO3

aspartate

In vertebrates, 2,3,5 catalyzedby trifunctional enzyme,6,7 catalyzed by bifunctional enzyme.

Pathways from IMP to AMP and GMP

G-1: IMP dehydrogenaseG-2: XMP aminaseA-1: adenylosuccinate synthetaseA-2: adenylosuccinate lyase

Note: GTP used to make AMP, ATP used to make GMP.Also, feedback inhibition byAMP and GMP.

Pathways 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.

Allosteric regulation of purine de novo synthesis

Purine degredation

AMP deamination in muscle, hydrolysis in other tissues.Xanthine oxidase:contains FAD, molybdenum, and non-heme iron.

In primates, uric acid is the end product, which is excreted.

Purine degredation in other animals

Clinical disorders of purine metabolism

Excessive accumulation of uric acid: Gout

The three defects shown each result in elevated de novo purine biosynthesis

Common treatment for gout: allopurinol

Allopurinol is an analogue of hypoxanthine that strongly inhibits xanthine oxidase. Xanthine and hypoxanthine, which are soluble, are accumulated and excreted.

Diseases of purine metabolism (continued)

Lesch-Nyhan Syndrome: Severe HGPRT deficiencyIn addition to symptoms of gout, patients display severe behavioral disorders, 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): 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 can’t replicate their DNA, and thus can’t mount an immune response.

De 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.

2 ATP + HCO3- + Glutamine + H2O

CO

O PO3-2

NH2

Carbamoyl Phosphate

NH2

CNH

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

HN

CNH

CH

CH2

C

COOO

O

Dihydroorotate

HN

CNH

C

CHC

COOO

O

Orotate

HN

CN

C

CHC

COOO

O

HH

CH2

OH OH

H HO

O2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HO

O2-O3P

Uridine Monophosphate(UMP)

2 ADP +Glutamate + Pi

CarbamoylPhosphateSynthetase II

AspartateTranscarbamoylase(ATCase)

Aspartate

Pi

H2O

Dihydroorotase

Quinone

ReducedQuinone

DihydroorotateDehydrogenase

PRPP PPi

Orotate PhosphoribosylTransferase

CO2

OMP Decarboxylase

Pyrimidine Synthesis

De novo synthesis of pyrimidines

1: carbamyl phosphate synthase2: aspartate transcarbamylase3: dihydroorotase4: dihydroorotate DH5: orotate phosphoribosyl tranferase6: orotidylate decarboxylase7: UMP kinase8: NDK9: CTP synthetase

CAD=1,2,35 +6=single protein

Regulation of pyrimidine de novo synthesis

Catabolism of pyrimidines

Overview of dNTP biosynthesis

One enzyme, ribonucleotide reductase,reduces all four ribonucleotides to theirdeoxyribo derivitives.

A free radical mechanism is involvedin the ribonucleotide reductasereaction.

There are three classes of ribonucleotidereductase enzymes in nature:Class I: tyrosine radical, uses NDPClass II: adenosylcobalamin. uses NTPs

(cyanobacteria, some bacteria,Euglena).

Class III: SAM and Fe-S to generateradical, uses NTPs.(anaerobes and fac. anaerobes).

Structure of rNDP reductase (E. coli, ClassI)

Proposed mechanism for rNDP reductase

Proposed reaction mechanism for ribonucleotide reductase

Sources of reducing power for rNDP reductase

Biological activities of thioredoxin

Regulation of activities of mammalian rNDP reductase

Salvage and de novo pathways to thymine nucleotides

Substrate recvognition by dUTPase

Relationship between thymidylate synthase and enzymes of tetrahydrofolate metabolism

Catalytic mechanism of thymidylate synthase

Regeneration of N5, N10-methylenetetrahydrofolate

Biosynthesis of NAD+ and NADP+

Biosynthesis of CoA from pantothenate

Proposed reaction mechanism for FGAM synthetase

The 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)

Proposed catalytic mechanism for OMP decarboxylase

Reactions catalyzed by eukaryotic dihydroorotate dehydrogenase

Nitrogenous BasesNitrogenous Bases

Planar, aromatic, and heterocyclicPlanar, aromatic, and heterocyclicDerived from Derived from purinepurine or or pyrimidinepyrimidineNumbering of bases is “unprimedNumbering of bases is “unprimed””

Nitrogenous BasesNitrogenous BasesPurines

Pyrimidines

N1: Aspartate AmineC2, C8: FormateN3, N9: GlutamineC4, C5, N7: GlycineC6: Bicarbonate Ion

Nucleotide MetabolismNucleotide MetabolismPURINE RIBONUCLEOTIDES: formed PURINE RIBONUCLEOTIDES: formed de novode novo

i.e., purines are i.e., purines are notnot initially synthesized as free bases initially synthesized as free basesFirst purine derivative formed is Inosine Mono-First purine derivative formed is Inosine Mono-

phosphate (IMP)phosphate (IMP)The purine base is The purine base is hypoxanthinehypoxanthineAMP and GMP are formed from IMPAMP and GMP are formed from IMP

Purine NucleotidesPurine Nucleotides

Get broken down into Uric Acid (a Get broken down into Uric Acid (a purine)purine)

Purine Nucleotide Purine Nucleotide SynthesisSynthesis

ATP is involved in 6 steps and an additional ATP is ATP is involved in 6 steps and an additional ATP is needed to form the first molecule (R5P)needed to form the first molecule (R5P)

PRPP in the first step of Purine synthesis is also a PRPP in the first step of Purine synthesis is also a precursor for Pyrimidine Synthesis, His and Trp synthesisprecursor for Pyrimidine Synthesis, His and Trp synthesis

Role of ATP in first step is unique– group transfer rather Role of ATP in first step is unique– group transfer rather than couplingthan coupling

In second step, CIn second step, C11 notation changes from notation changes from to to (anomers (anomers specifying OH positioning on Cspecifying OH positioning on C11 with respect to C with respect to C44 group) group)

In step 3, PPIn step 3, PPii is hydrolyzed to 2P is hydrolyzed to 2Pii (irreversible, (irreversible, “committing” step)“committing” step)

Hydrolyzing a phosphate from ATP is relatively easyHydrolyzing a phosphate from ATP is relatively easy G°’= -30.5 kJ/molG°’= -30.5 kJ/mol

If endergonic reaction released energy into cell as heat If endergonic reaction released energy into cell as heat energy, wouldn’t be usefulenergy, wouldn’t be useful

Must be coupled to an exergonic reactionMust be coupled to an exergonic reactionWhen ATP is a reactantWhen ATP is a reactant::

Part of the ATP can be transferred to an acceptor: PPart of the ATP can be transferred to an acceptor: Pii, PP, PPii, , adenyl, adenyl, or adenosinyl group in or adenosinyl group in transferasetransferase reaction reaction

ORORATP hydrolysis can drive an otherwise unfavorable reactionATP hydrolysis can drive an otherwise unfavorable reaction

((synthetase; “energasesynthetase; “energase)”)”

Coupling of ReactionsCoupling of Reactions

Purine Biosynthetic Purine Biosynthetic PathwayPathway

Coupling of some reactions on pathway organizes and Coupling of some reactions on pathway organizes and controls processing of substrates to products in each controls processing of substrates to products in each

stepstepIncreases overall rate of pathway and protects Increases overall rate of pathway and protects

intermediates from degradationintermediates from degradationIn animals, IMP synthesis pathway is coupledIn animals, IMP synthesis pathway is coupled::

Reactions 3, 4, 6Reactions 3, 4, 6Reactions 7, 8Reactions 7, 8Reactions 10, 11Reactions 10, 11

IMP Conversion to AMPIMP Conversion to AMP

IMP Conversion to GMPIMP Conversion to GMP

Regulatory Control of Purine Regulatory Control of Purine Nucleotide BiosynthesisNucleotide Biosynthesis

GTP is involved in AMP synthesis and ATP is GTP is involved in AMP synthesis and ATP is involved in GMP synthesis (reciprocal control of involved in GMP synthesis (reciprocal control of

production)production)PRPP is a biosynthetically “central” molecule PRPP is a biosynthetically “central” molecule

(why?)(why?)ADP/GDP levels – negative feedback on Ribose Phosphate ADP/GDP levels – negative feedback on Ribose Phosphate

PyrophosphokinasePyrophosphokinase Amidophosphoribosyl transferase is activated by PRPP Amidophosphoribosyl transferase is activated by PRPP

levelslevelsAPRT activity has negative feedback at two sitesAPRT activity has negative feedback at two sites

ATP, ADP, AMP bound at one siteATP, ADP, AMP bound at one siteGTP,GDP AND GMP bound at the other siteGTP,GDP AND GMP bound at the other site

Rate of AMP production increases with increasing Rate of AMP production increases with increasing concentrations of GTP; rate of GMP production concentrations of GTP; rate of GMP production

increases with increasing concentrations of ATPincreases with increasing concentrations of ATP

Regulatory Control of Purine Regulatory Control of Purine BiosynthesisBiosynthesis

At level of IMP productionAt level of IMP production::Independent controlIndependent controlSynergistic controlSynergistic controlFeedforward activation by PRPPFeedforward activation by PRPP

Below level of IMP productionBelow level of IMP productionReciprocal controlReciprocal control

Total amounts of purine nucleotides Total amounts of purine nucleotides controlledcontrolled

Relative amounts of ATP, GTP controlledRelative amounts of ATP, GTP controlled

Purine Catabolism and Purine Catabolism and SalvageSalvage

All purine degradation in animals leads to All purine degradation in animals leads to uric aciduric acidIngested nucleic acids are degraded by pancreatic Ingested nucleic acids are degraded by pancreatic

nucleases, and intestinal phosphodiesterases in the nucleases, and intestinal phosphodiesterases in the intestineintestine

Group-specific nucleotidases and non-specific Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosidesphosphatases degrade nucleotides into nucleosides

Direct absorption of nucleosidesDirect absorption of nucleosides Further degradationFurther degradation

Nucleoside + HNucleoside + H22O O base + ribose (nucleosidase) base + ribose (nucleosidase)

Nucleoside + PNucleoside + Pii base + r-1-phosphate (n. phosphorylase) base + r-1-phosphate (n. phosphorylase)

NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETEDAND EXCRETED..

Intracellular Purine Intracellular Purine CatabolismCatabolism

Nucleotides broken into nucleosides by Nucleotides broken into nucleosides by action of 5’-nucleotidase (hydrolysis action of 5’-nucleotidase (hydrolysis

reactions)reactions)Purine nucleoside phosphorylase (PNP)Purine nucleoside phosphorylase (PNP)

Inosine Inosine Hypoxanthine HypoxanthineXanthosine Xanthosine Xanthine XanthineGuanosine Guanosine Guanine GuanineRibose-1-phosphate splits offRibose-1-phosphate splits off

Can be isomerized to ribose-5-phosphateCan be isomerized to ribose-5-phosphate

Adenosine is deaminated to Inosine (ADA)Adenosine is deaminated to Inosine (ADA)

Intracellular Purine Intracellular Purine CatabolismCatabolism

Xanthine is the point of convergence for Xanthine is the point of convergence for the metabolism of the purine basesthe metabolism of the purine bases

Xanthine Xanthine Uric acid Uric acidXanthine oxidase catalyzes two reactionsXanthine oxidase catalyzes two reactions

Purine ribonucleotide degradation Purine ribonucleotide degradation pathway is same for purine pathway is same for purine

deoxyribonucleotidesdeoxyribonucleotides

Adenosine DegradationAdenosine Degradation

Xanthosine DegradationXanthosine Degradation

• Ribose sugar gets recycled )Ribose-1-Phosphate R-5-P ( – can be incorporated into PRPP )efficiency(• Hypoxanthine is converted to Xanthine by Xanthine Oxidase• Guanine is converted to Xanthine by Guanine Deaminase• Xanthine gets converted to Uric Acid by Xanthine Oxidase

Xanthine OxidaseXanthine Oxidase

A homodimeric proteinA homodimeric proteinContains electron transfer proteinsContains electron transfer proteins

FADFADMo-pterin complex in +4 or +6 stateMo-pterin complex in +4 or +6 state Two 2Fe-2S clustersTwo 2Fe-2S clusters

Transfers electrons to OTransfers electrons to O22 H H22OO22

HH22OO22 is toxic is toxic Disproportionated to HDisproportionated to H22O and OO and O22 by catalase by catalase

AMP + HAMP + H22O O IMP + NH IMP + NH44++ (AMP Deaminase)(AMP Deaminase)

IMP + Aspartate + GTP IMP + Aspartate + GTP AMP + Fumarate + GDP AMP + Fumarate + GDP + P+ Pii (Adenylosuccinate Synthetase)(Adenylosuccinate Synthetase)

COMBINE THE TWO REACTIONSCOMBINE THE TWO REACTIONS::

Aspartate + HAspartate + H22O + GTP O + GTP Fumarate + GDP + P Fumarate + GDP + Pii + + NHNH44

++

The overall result of combining reactions is deamination of The overall result of combining reactions is deamination of Aspartate to Aspartate to Fumarate at the expense of a GTPFumarate at the expense of a GTP

THE PURINE NUCLEOTIDE THE PURINE NUCLEOTIDE CYCLECYCLE

Purine Nucleotide CyclePurine Nucleotide Cycle

In-Class Question: Why is the purine In-Class Question: Why is the purine nucleotide cycle important in muscle nucleotide cycle important in muscle metabolism during a burst of activitymetabolism during a burst of activity??

Adenosine DeaminaseAdenosine Deaminase

CHIME Exercise: 2ADACHIME Exercise: 2ADAEnzyme catalyzing deamination of Adenosine to Enzyme catalyzing deamination of Adenosine to

InosineInosine // barrel domain structure barrel domain structure

““TIM Barrel” – central barrel structure with 8 TIM Barrel” – central barrel structure with 8 twisted parallel twisted parallel -strands connected by 8 -strands connected by 8 --

helical loopshelical loopsActive site is at bottom of funnel-shaped pocket Active site is at bottom of funnel-shaped pocket

formed by loopsformed by loopsFound in all glycolytic enzymesFound in all glycolytic enzymesFound in proteins that bind and transport Found in proteins that bind and transport

metabolitesmetabolites

Uric Acid ExcretionUric Acid Excretion

Humans – excreted into urine as Humans – excreted into urine as insoluble crystalsinsoluble crystals

Birds, terrestrial reptiles, some Birds, terrestrial reptiles, some insects – excrete isoluble crystals in insects – excrete isoluble crystals in

paste form (conserve water)paste form (conserve water)Others – further modificationOthers – further modification: :

Uric Acid Uric Acid Allantoin Allantoin Allantoic Acid Allantoic Acid Urea Urea AmmoniaAmmonia

Purine Purine SalvageSalvage

Adenine phosphoribosyl transferase (APRT)Adenine phosphoribosyl transferase (APRT)Adenine + PRPP Adenine + PRPP AMP + PP AMP + PPii

Hypoxanthine-Guanine phosphoribosyl Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT)transferase (HGPRT)

Hypoxanthine + PRPP Hypoxanthine + PRPP IMP + PP IMP + PPii

Guanine + PRPP Guanine + PRPP GMP + PP GMP + PPii

((NOTE: THESE ARE ALL NOTE: THESE ARE ALL REVERSIBLEREVERSIBLE REACTIONS REACTIONS))

AMP,IMP,GMP do not need to be AMP,IMP,GMP do not need to be resynthesized resynthesized de novode novo! !

A CASE STUDY : GOUTA CASE STUDY : GOUTA 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A

PAINFUL AND SWOLLEN RIGHT GREAT TOE. ON THE PAINFUL AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT HE HAD EATEN A MEAL OF FRIED PREVIOUS NIGHT HE HAD EATEN A MEAL OF FRIED

LIVER AND ONIONS, AFTER WHICH HE MET WITH HIS LIVER AND ONIONS, AFTER WHICH HE MET WITH HIS POKER GROUP AND DRANK A NUMBER OF BEERSPOKER GROUP AND DRANK A NUMBER OF BEERS..

HE SAW HIS DOCTOR THAT MORNING, “GOUTY HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS” WAS DIAGNOSED, AND SOME TESTS ARTHRITIS” WAS DIAGNOSED, AND SOME TESTS

WERE ORDERED. HIS SERUM URIC ACID LEVEL WAS WERE ORDERED. HIS SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL < 7.0 mg/dL)ELEVATED AT 8.0 mg/dL (NL < 7.0 mg/dL)..

THE MAN RECALLED THAT HIS FATHER AND HIS THE MAN RECALLED THAT HIS FATHER AND HIS GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS, GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS,

OFTEN COMPLAINED OF JOINT PAIN AND SWELLING OFTEN COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEETIN THEIR FEET..

A CASE STUDY : GOUTA CASE STUDY : GOUTTHE DOCTOR RECOMMENDED THAT THE THE DOCTOR RECOMMENDED THAT THE

MAN USE NSAIDS FOR PAIN AND SWELLING, MAN USE NSAIDS FOR PAIN AND SWELLING, INCREASE HIS FLUID INTAKE (BUT NOT WITH INCREASE HIS FLUID INTAKE (BUT NOT WITH

ALCOHOL) AND REST AND ELEVATE HIS ALCOHOL) AND REST AND ELEVATE HIS FOOT. HE ALSO PRESCRIBED ALLOPURINOLFOOT. HE ALSO PRESCRIBED ALLOPURINOL . .

A FEW DAYS LATER THE CONDITION HAD A FEW DAYS LATER THE CONDITION HAD RESOLVED AND ALLOPURINOL HAD BEEN RESOLVED AND ALLOPURINOL HAD BEEN

STOPPED. A REPEAT URIC ACID LEVEL WAS STOPPED. A REPEAT URIC ACID LEVEL WAS OBTAINED (7.1 mg/dL). THE DOCTOR GAVE OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE MAN SOME ADVICE REGARDING LIFE THE MAN SOME ADVICE REGARDING LIFE

STYLE CHANGESSTYLE CHANGES..

GoutGoutImpaired excretion or overproduction Impaired excretion or overproduction

of uric acidof uric acidUric acid crystals precipitate into Uric acid crystals precipitate into

joints (Gouty Arthritis), kidneys, joints (Gouty Arthritis), kidneys, ureters (stones)ureters (stones)

Lead impairs uric acid excretion – lead Lead impairs uric acid excretion – lead poisoning from pewter drinking poisoning from pewter drinking

gobletsgobletsFall of Roman EmpireFall of Roman Empire??

Xanthine oxidase inhibitors inhibit Xanthine oxidase inhibitors inhibit production of uric acid, and treat goutproduction of uric acid, and treat gout

Allopurinol treatment – hypoxanthine Allopurinol treatment – hypoxanthine analog that binds to Xanthine Oxidase analog that binds to Xanthine Oxidase

to decrease uric acid productionto decrease uric acid production

ALLOPURINOL IS A XANTHINE ALLOPURINOL IS A XANTHINE OXIDASE INHIBITOROXIDASE INHIBITOR

A SUBSTRATE ANALOG IS CONVERTED TO A SUBSTRATE ANALOG IS CONVERTED TO AN INHIBITOR, IN THIS CASE A “SUICIDE-AN INHIBITOR, IN THIS CASE A “SUICIDE-

INHIBITORINHIBITOR””

Lesch-Nyhan SyndromeLesch-Nyhan SyndromeA defect in production or activity ofA defect in production or activity of

HGPRTHGPRT Causes increased level of Hypoxanthine Causes increased level of Hypoxanthine

and Guanine (and Guanine ( in degradation to uric in degradation to uric acid)acid)

Also,PRPP accumulatesAlso,PRPP accumulates stimulates production of purine stimulates production of purine

nucleotides (and thereby increases their nucleotides (and thereby increases their degradation)degradation)

Causes gout-like symptoms, but also Causes gout-like symptoms, but also neurological symptoms neurological symptoms spasticity, spasticity,

aggressiveness, self-mutilationaggressiveness, self-mutilationFirst neuropsychiatric abnormality that First neuropsychiatric abnormality that

was attributed to a single enzymewas attributed to a single enzyme

Purine AutismPurine Autism25%25% of autistic patients may of autistic patients may

overproduce purinesoverproduce purinesTo diagnose, must test urine To diagnose, must test urine

over 24 hoursover 24 hoursBiochemical findings from this Biochemical findings from this

test disappear in adolescencetest disappear in adolescenceMust obtain urine specimen in Must obtain urine specimen in

infancy, but it’s difficult to doinfancy, but it’s difficult to do!!Pink urine due to uric acid crystals Pink urine due to uric acid crystals

may be seen in diapersmay be seen in diapers

Pyrimidine Ribonucleotide Pyrimidine Ribonucleotide SynthesisSynthesis

Uridine Monophosphate (UMP) is Uridine Monophosphate (UMP) is synthesized firstsynthesized first

CTP is synthesized from UMPCTP is synthesized from UMPPyrimidine ring synthesis completed Pyrimidine ring synthesis completed

first; then attached to ribose-5-first; then attached to ribose-5-phosphatephosphate

N1, C4, C5, C6 : AspartateC2 : HCO3

-

N3 : Glutamine amide Nitrogen

UMP Synthesis OverviewUMP Synthesis Overview22 ATPs needed: both used in first stepATPs needed: both used in first step

One transfers phosphate, the other is hydrolyzed to One transfers phosphate, the other is hydrolyzed to ADP and PiADP and Pi

22 condensation rxns: form carbamoyl aspartate condensation rxns: form carbamoyl aspartate and dihydroorotate (intramolecular)and dihydroorotate (intramolecular)

Dihydroorotate dehydrogenase is an Dihydroorotate dehydrogenase is an intra-intra-mitochondrial mitochondrial enzyme; oxidizing power comes enzyme; oxidizing power comes

from quinone reductionfrom quinone reductionAttachment of base to ribose ring is catalyzed by Attachment of base to ribose ring is catalyzed by

OPRT; OPRT; PRPP provides ribose-5-PPRPP provides ribose-5-PPPPPii splits off PRPP – irreversible splits off PRPP – irreversible

Channeling: enzymes 1, 2, and 3 on same chain; Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on same chain5 and 6 on same chain

2 ATP + HCO3- + Glutamine + H2O

CO

O PO3-2

NH2

Carbamoyl Phosphate

NH2

CNH

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

HN

CNH

CH

CH2

C

COOO

O

Dihydroorotate

HN

CNH

C

CHC

COOO

O

Orotate

HN

CN

C

CHC

COOO

O

HH

CH2

OH OH

H HO

O2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HO

O2-O3P

Uridine Monophosphate(UMP)

2 ADP +Glutamate + Pi

CarbamoylPhosphateSynthetase II

AspartateTranscarbamoylase(ATCase)

Aspartate

Pi

H2O

Dihydroorotase

Quinone

ReducedQuinone

DihydroorotateDehydrogenase

PRPP PPi

Orotate PhosphoribosylTransferase

CO2

OMP Decarboxylase

Pyrimidine Synthesis

UMP UMP UTP and CTP UTP and CTP

Nucleoside monophosphate kinase Nucleoside monophosphate kinase catalyzes transfer of Pcatalyzes transfer of Pii to UMP to form to UMP to form

UDP; nucleoside diphosphate kinase UDP; nucleoside diphosphate kinase catalyzes transfer of Pcatalyzes transfer of Pii from ATP to UDP from ATP to UDP

to form UTPto form UTP

CTP formed from UTP via CTP formed from UTP via CTP SynthetaseCTP Synthetase driven by ATP hydrolysisdriven by ATP hydrolysis

Glutamine provides amide nitrogen for Glutamine provides amide nitrogen for CC44

OMP DECARBOXYLASE : THE OMP DECARBOXYLASE : THE MOST CATALYTICALLY MOST CATALYTICALLY PROFICIENT ENZYMEPROFICIENT ENZYME

FINAL REACTION OF PYRIMIDINE PATHWAYFINAL REACTION OF PYRIMIDINE PATHWAYANOTHER MECHANISM FOR ANOTHER MECHANISM FOR

DECARBOXYLATIONDECARBOXYLATIONA CARBANION INTERMEDIATE (UNSTABLE)A CARBANION INTERMEDIATE (UNSTABLE)

MUST BE STABILIZEDMUST BE STABILIZEDBUT NO COFACTORS ARE NEEDEDBUT NO COFACTORS ARE NEEDED!!

SOME OF THE BINDING ENERGY BETWEEN SOME OF THE BINDING ENERGY BETWEEN OMP AND THE ACTIVE SITE IS USED TO OMP AND THE ACTIVE SITE IS USED TO

STABILIZE THE TRANSITION STATESTABILIZE THE TRANSITION STATE““PREFERENTIAL TRANSITION STATE BINDINGPREFERENTIAL TRANSITION STATE BINDING””

Regulatory Control of Regulatory Control of Pyrimidine SynthesisPyrimidine Synthesis

Differs between bacteria and animalsDiffers between bacteria and animalsBacteria – regulation at ATCase rxnBacteria – regulation at ATCase rxn

AnimalsAnimals – regulation at carbamoyl phosphate – regulation at carbamoyl phosphate synthetase IIsynthetase II

UDP and UTP inhibit enzyme; ATP and PRPP activate UDP and UTP inhibit enzyme; ATP and PRPP activate itit

UMP and CMP competitively inhibit OMP UMP and CMP competitively inhibit OMP DecarboxylaseDecarboxylase

**Purine synthesis inhibited by ADP and GDP at Purine synthesis inhibited by ADP and GDP at ribose phosphate pyrophosphokinase step, ribose phosphate pyrophosphokinase step, controlling level of PRPP controlling level of PRPP also regulates also regulates

pyrimidinespyrimidines

Orotic AciduriaOrotic AciduriaCaused by defect in protein chain with Caused by defect in protein chain with

enzyme activities of last two steps of enzyme activities of last two steps of pyrimidine synthesispyrimidine synthesis

Increased excretion of orotic acid in Increased excretion of orotic acid in urineurine

Symptoms: retarded growth; severe Symptoms: retarded growth; severe anemiaanemia

Only known inherited defect in this Only known inherited defect in this pathway pathway (all others would be lethal to (all others would be lethal to

fetus)fetus)Treat with uridine/cytidineTreat with uridine/cytidine IN-CLASS QUESTION: HOW DOES URIDINE IN-CLASS QUESTION: HOW DOES URIDINE

AND CYTIDINE ADMINISTRATION WORK TO AND CYTIDINE ADMINISTRATION WORK TO TREAT OROTICACIDURIATREAT OROTICACIDURIA??

Degradation of Degradation of PyrimidinesPyrimidines

CMP and UMP degraded to bases CMP and UMP degraded to bases similarly to purinessimilarly to purines

DephosphorylationDephosphorylationDeaminationDeaminationGlycosidic bond cleavageGlycosidic bond cleavage

Uracil reduced in liver, forming Uracil reduced in liver, forming --alaninealanine

Converted to malonyl-CoA Converted to malonyl-CoA fatty acid fatty acid synthesis for energy metabolismsynthesis for energy metabolism

Deoxyribonucleotide Deoxyribonucleotide FormationFormation

Purine/Pyrimidine degradation are the Purine/Pyrimidine degradation are the same for ribonucleotides and same for ribonucleotides and

deoxyribonucleotidesdeoxyribonucleotides

Biosynthetic pathways are only for Biosynthetic pathways are only for ribonucleotidesribonucleotides

Deoxyribonucleotides are synthesized Deoxyribonucleotides are synthesized from corresponding ribonucleotidesfrom corresponding ribonucleotides

DNA vs. RNA: REVIEWDNA vs. RNA: REVIEW

DNA composed of deoxyribonucleotidesDNA composed of deoxyribonucleotides

Ribose sugar in DNA lacks hydroxyl group Ribose sugar in DNA lacks hydroxyl group at 2’ Carbonat 2’ Carbon

Uracil doesn’t (normally) appear in DNAUracil doesn’t (normally) appear in DNAThymine (5-methyluracil) appears insteadThymine (5-methyluracil) appears instead

Formation of Formation of DeoxyribonucleotidesDeoxyribonucleotides

Reduction of 2’ carbon done via a Reduction of 2’ carbon done via a free free radical mechanismradical mechanism catalyzed by catalyzed by

“Ribonucleotide Reductases“Ribonucleotide Reductases ” ”

E. coli E. coli RNR reduces ribonucleoside RNR reduces ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (NDPs) to deoxyribonucleoside

diphosphates (dNDPs)diphosphates (dNDPs)Two subunits: R1 and R2Two subunits: R1 and R2

A Heterotetramer: (R1)A Heterotetramer: (R1)22 and (R2) and (R2)2 2 in vitroin vitro

RIBONUCLEOTIDE REDUCTASERIBONUCLEOTIDE REDUCTASE

R1 SUBUNITR1 SUBUNITSpecificity SiteSpecificity SiteHexamerization siteHexamerization siteActivity SiteActivity SiteFive redox-active –SH groups from cysteinesFive redox-active –SH groups from cysteines

R2 SUBUNITR2 SUBUNITTyr 122 radicalTyr 122 radicalBinuclear Fe(III) complexBinuclear Fe(III) complex

Chime ExerciseChime Exercise

E. coli E. coli Ribonucleotide Ribonucleotide ReductaseReductase::

3R1R and 4R1R: R1 subunit3R1R and 4R1R: R1 subunit

1RIB and 1AV8: R2 subunit1RIB and 1AV8: R2 subunit

Ribonucleotide Reductase Ribonucleotide Reductase R2 SubunitR2 Subunit

Fe prosthetic group– binuclear, with each Fe prosthetic group– binuclear, with each Fe Fe octahedrallyoctahedrally coordinated coordinated

Fe’s are bridged by OFe’s are bridged by O-2-2 and carboxyl gp of Glu and carboxyl gp of Glu 115115

Tyr 122 is close to the Fe(III) complex Tyr 122 is close to the Fe(III) complex stabilization of a tyrosyl free-radicalstabilization of a tyrosyl free-radical

During the overall process, a pair of –SH During the overall process, a pair of –SH groups provide the reducing equivalentsgroups provide the reducing equivalents

A protein disulfide group is formedA protein disulfide group is formedGets reduced by two other sulfhydryl gps of Gets reduced by two other sulfhydryl gps of

Cys residues in R1Cys residues in R1

Mechanism of Ribonucleotide Mechanism of Ribonucleotide Reductase ReactionReductase Reaction

Free RadicalFree RadicalInvolvement of multiple –SH groupsInvolvement of multiple –SH groupsRR is left with a disulfide group that RR is left with a disulfide group that

must be reduced to return to the must be reduced to return to the original enzymeoriginal enzyme

RIBONUCLEOTIDE REDUCTASERIBONUCLEOTIDE REDUCTASE

ACTIVITY IS RESPONSIVE TO LEVEL OF ACTIVITY IS RESPONSIVE TO LEVEL OF CELLULAR NUCLEOTIDESCELLULAR NUCLEOTIDES::

ATP ACTIVATES REDUCTION OFATP ACTIVATES REDUCTION OFCDPCDPUDPUDP

dTTPdTTP INDUCES GDP REDUCTIONINDUCES GDP REDUCTIONINHIBITS REDUCTION OF CDP. UDPINHIBITS REDUCTION OF CDP. UDP

dATP INHIBITS REDUCTION OF ALL NUCLEOTIDESdATP INHIBITS REDUCTION OF ALL NUCLEOTIDESdGTPdGTP

STIMULATES ADP REDUCTIONSTIMULATES ADP REDUCTIONINHIBITS CDP,UDP,GDP REDUCTIONINHIBITS CDP,UDP,GDP REDUCTION

RIBONUCLEOTIDE REDUCTASERIBONUCLEOTIDE REDUCTASE

CATALYTIC ACTIVITY VARIES WITH STATE OF CATALYTIC ACTIVITY VARIES WITH STATE OF OLIGOMERIZATIONOLIGOMERIZATION::

WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE OF R1 (CATALYTICALLY INACTIVE MONOMER)SITE OF R1 (CATALYTICALLY INACTIVE MONOMER)

CATALYTICALLY ACTIVE (R1)CATALYTICALLY ACTIVE (R1)22

WHEN dATP OR ATP BIND TO ACTIVITY SITE OF WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERSDIMERS

TETRAMER FORMATIONTETRAMER FORMATION((R1R1))4a4a (ACTIVE STATE) == (R1) (ACTIVE STATE) == (R1)4b4b (INACTIVE) (INACTIVE)

WHEN ATP BINDS TO HEXAMERIZATION SITEWHEN ATP BINDS TO HEXAMERIZATION SITE CATALYTICALLY ACTIVE HEXAMERS (R1)CATALYTICALLY ACTIVE HEXAMERS (R1)66

ThioredoxinThioredoxinPhysiologic reducing agent of RNRPhysiologic reducing agent of RNRCys pair can swap H atoms with disulfide Cys pair can swap H atoms with disulfide

formed formed regenerate original enzymeregenerate original enzymeThioredoxin gets oxidized to disulfideThioredoxin gets oxidized to disulfide

Oxidized Thioredoxin gets reduced by thioredoxin reductase mediatedby NADPH (final electron acceptor)

Thymine FormationThymine Formation

Formed by methylating deoxyuridine Formed by methylating deoxyuridine monophosphate (dUMP)monophosphate (dUMP)

UTP needed for RNA production, but UTP needed for RNA production, but dUTP not needed for DNAdUTP not needed for DNA

If dUTP produced excessively, would cause If dUTP produced excessively, would cause substitution errors (dUTP for dTTP)substitution errors (dUTP for dTTP)

dUTP hydrolyzed by dUTP dUTP hydrolyzed by dUTP diphosphohydrolase to dUMP diphosphohydrolase to dUMP

methylated at C5 to form dTMPmethylated at C5 to form dTMP rephosphorylate to form dTTPrephosphorylate to form dTTP

Chime ExerciseChime Exercise

1DUD: dUTPase1DUD: dUTPase

Tetrahydrofolate (THF)Tetrahydrofolate (THF)

Methylation of dUMP catalyzed by Methylation of dUMP catalyzed by thymidylate synthasethymidylate synthase

Cofactor: NCofactor: N55,N,N1010-methylene THF-methylene THFOxidized to dihydrofolateOxidized to dihydrofolate

Only known rxn where net oxidation Only known rxn where net oxidation state of THF changesstate of THF changes

THF RegenerationTHF Regeneration::DHF + NADPH + HDHF + NADPH + H++ THF + NADP THF + NADP++ (enzyme: dihydrofolate (enzyme: dihydrofolate

reductase)reductase)

THF + Serine THF + Serine N N55,N,N1010-methylene-THF + Glycine-methylene-THF + Glycine ((enzyme: serine hydroxymethyl transferaseenzyme: serine hydroxymethyl transferase))

Anti-Folate DrugsAnti-Folate Drugs

Cancer cells consume dTMP quickly for Cancer cells consume dTMP quickly for DNA replicationDNA replication

Interfere with thymidylate synthase rxn to Interfere with thymidylate synthase rxn to decrease dTMP productiondecrease dTMP production

((fluorodeoxyuridylate – irreversible inhibitorfluorodeoxyuridylate – irreversible inhibitor – ) – )also also affects rapidly growing normal cells (hair follicles, affects rapidly growing normal cells (hair follicles, bone marrow, immune system, intestinal mucosa)bone marrow, immune system, intestinal mucosa)

Dihydrofolate reductase step can be Dihydrofolate reductase step can be stopped competitively (DHF analogs)stopped competitively (DHF analogs)

Anti-Folates: Aminopterin, methotrexate, Anti-Folates: Aminopterin, methotrexate, trimethoprimtrimethoprim

IN-CLASS QUESTIONIN-CLASS QUESTION

IN von GIERKE’S DISEASE, OVERPRO- IN von GIERKE’S DISEASE, OVERPRO- DUCTION OF URIC ACID OCCURS. THIS DUCTION OF URIC ACID OCCURS. THIS

DISEASE IS CAUSED BY A DEFICIENCY DISEASE IS CAUSED BY A DEFICIENCY OF GLUCOSE-6-PHOSPHATASEOF GLUCOSE-6-PHOSPHATASE..

EXPLAIN THE BIOCHEMICAL EVENTS THAT EXPLAIN THE BIOCHEMICAL EVENTS THAT LEAD TO INCREASED URIC ACID LEAD TO INCREASED URIC ACID

PRODUCTIONPRODUCTION??WHY DOES HYPOGLYCEMIA OCCUR IN THIS WHY DOES HYPOGLYCEMIA OCCUR IN THIS

DISEASEDISEASE??WHY IS THE LIVER ENLARGEDWHY IS THE LIVER ENLARGED??

ADENOSINE DEAMINASE ADENOSINE DEAMINASE DEFICIENCYDEFICIENCY

IN PURINE DEGRADATION, ADENOSINE IN PURINE DEGRADATION, ADENOSINE INOSINEINOSINE

ENZYME IS ADAENZYME IS ADAADA DEFICIENCY RESULTS IN SCIDADA DEFICIENCY RESULTS IN SCID

““SEVERE COMBINED IMMUNODEFICIENCYSEVERE COMBINED IMMUNODEFICIENCY””

SELECTIVELY KILLS LYMPHOCYTESSELECTIVELY KILLS LYMPHOCYTESBOTH B- AND T-CELLSBOTH B- AND T-CELLSMEDIATE MUCH OF IMMUNE RESPONSEMEDIATE MUCH OF IMMUNE RESPONSE

ALL KNOWN ADA MUTANTS STRUCTURALLY ALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB ACTIVE SITEPERTURB ACTIVE SITE

ADA DEFICIENCYADA DEFICIENCY

IN-CLASS QUESTION: EXPLAIN THE IN-CLASS QUESTION: EXPLAIN THE BIOCHEMISTRY THAT RESULTS WHEN A BIOCHEMISTRY THAT RESULTS WHEN A

PERSON HAS ADA DEFICIENCYPERSON HAS ADA DEFICIENCY

((HINT: LYMPHOID TISSUE IS VERY ACTIVE IN HINT: LYMPHOID TISSUE IS VERY ACTIVE IN DEOXYADENOSINE PHOSPHORYLATIONDEOXYADENOSINE PHOSPHORYLATION))

ADA DEFICIENCYADA DEFICIENCYONE OF FIRST DISEASES TO BE TREATED ONE OF FIRST DISEASES TO BE TREATED

WITH GENE THERAPYWITH GENE THERAPY

ADA GENE INSERTED INTO LYMPHOCYTES; ADA GENE INSERTED INTO LYMPHOCYTES; THEN LYMPHOCYTES RETURNED TO PATIENTTHEN LYMPHOCYTES RETURNED TO PATIENT

PEG-ADA TREATMENTSPEG-ADA TREATMENTSACTIVITY LASTS 1-2 WEEKSACTIVITY LASTS 1-2 WEEKS

SugarsSugars

Pentoses (5-C sugars)Pentoses (5-C sugars)Numbering of sugars is “primedNumbering of sugars is “primed””

SugarsSugars

D-Ribose and 2’-DeoxyriboseD-Ribose and 2’-Deoxyribose

*Lacks a 2’-OH group

NucleosidesNucleosides

Result from linking one of the sugars Result from linking one of the sugars with a purine or pyrimidine base with a purine or pyrimidine base through an N-glycosidic linkagethrough an N-glycosidic linkage

Purines bond to the C1’ carbon of the Purines bond to the C1’ carbon of the sugar at their N9 atomssugar at their N9 atoms

Pyrimidines bond to the C1’ carbon of Pyrimidines bond to the C1’ carbon of the sugar at their N1 atomsthe sugar at their N1 atoms

NucleosidesNucleosides

Phosphate GroupsPhosphate Groups

Mono-, di- or triphosphatesMono-, di- or triphosphates

Phosphates can be bonded to either Phosphates can be bonded to either C3 or C5 atoms of the sugarC3 or C5 atoms of the sugar

NucleotidesNucleotides

Result from linking one or more Result from linking one or more phosphates with a nucleoside onto the 5’ phosphates with a nucleoside onto the 5’

end of the molecule through esterificationend of the molecule through esterification

NucleotidesNucleotides

RNA (ribonucleic acid) is a polymer of RNA (ribonucleic acid) is a polymer of ribonucleotidesribonucleotides

DNA (deoxyribonucleic acid) is a DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotidespolymer of deoxyribonucleotides

Both deoxy- and ribonucleotides Both deoxy- and ribonucleotides contain Adenine, Guanine and contain Adenine, Guanine and

CytosineCytosineRibonucleotides contain UracilRibonucleotides contain UracilDeoxyribonucleotides contain ThymineDeoxyribonucleotides contain Thymine

NucleotidesNucleotides

Monomers for nucleic acid polymersMonomers for nucleic acid polymersNucleoside Triphosphates are Nucleoside Triphosphates are

important energy carriers (ATP, important energy carriers (ATP, GTP)GTP)

Important components of coenzymesImportant components of coenzymesFAD, NADFAD, NAD++ and Coenzyme A and Coenzyme A

Naming ConventionsNaming Conventions

NucleosidesNucleosides::Purine nucleosides end in “-sinePurine nucleosides end in “-sine ” ”

Adenosine, CytosineAdenosine, CytosinePyrimidine nucleosides end in “-dinePyrimidine nucleosides end in “-dine””

Thymidine, GuanidineThymidine, GuanidineNucleotidesNucleotides::

Start with the nucleoside name from Start with the nucleoside name from above and add “mono-”, “di-”, or above and add “mono-”, “di-”, or

“triphosphate“triphosphate””Adenosine Monophosphate, Guanidine Adenosine Monophosphate, Guanidine

Triphosphate, Deoxythymidine DiphosphateTriphosphate, Deoxythymidine Diphosphate

In-Class ActivitiesIn-Class Activities

Look at theLook at the Nucleotide StructuresNucleotide Structures

Take theTake the Nucleotide Identification QuizNucleotide Identification Quiz

Be prepared to identify some of these Be prepared to identify some of these structures on an exam. Learn some structures on an exam. Learn some “tricks” that help you to distinguish “tricks” that help you to distinguish

among the different structuresamong the different structures