Nucleotides:Nucleotides: Synthesis and Synthesis and DegradationDegradation

Nitrogenous BasesNitrogenous Bases

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

Nucleic Acid BasesNucleic Acid BasesPurines Pyrimidines

SugarsSugars

Pentoses (5-C sugars)Pentoses (5-C sugars)Numbering of sugars is “primed”Numbering 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 with Result from linking one of the sugars with a purine or pyrimidine base through an N-a purine or pyrimidine base through an N-glycosidic linkageglycosidic linkage

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

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

NucleosidesNucleosides

Phosphate GroupsPhosphate Groups

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

Phosphates can be bonded to either C3 or Phosphates can be bonded to either C3 or C5 atoms of the sugarC5 atoms of the sugar

NucleotidesNucleotides

Result from linking one or more phosphates Result from linking one or more phosphates with a nucleoside onto the 5’ end of the with a nucleoside onto the 5’ end of the molecule through esterificationmolecule through esterification

NucleotidesNucleotides

RNA (ribonucleic acid) is a polymer of RNA (ribonucleic acid) is a polymer of ribonucleotidesribonucleotidesDNA (deoxyribonucleic acid) is a polymer DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotidesof deoxyribonucleotidesBoth deoxy- and ribonucleotides contain Both deoxy- and ribonucleotides contain Adenine, Guanine and CytosineAdenine, Guanine and Cytosine– Ribonucleotides contain UracilRibonucleotides contain Uracil– Deoxyribonucleotides contain ThymineDeoxyribonucleotides contain Thymine

NucleotidesNucleotides

Monomers for nucleic acid polymersMonomers for nucleic acid polymersNucleoside Triphosphates are important Nucleoside Triphosphates are important energy carriers (ATP, GTP)energy carriers (ATP, GTP)Important components of coenzymesImportant components of coenzymes– FAD, NADFAD, NAD++ and Coenzyme A and Coenzyme A

Naming ConventionsNaming Conventions

Nucleosides:Nucleosides:– Purine nucleosides end in “-sine” Purine nucleosides end in “-sine”

Adenosine, GuanosineAdenosine, Guanosine– Pyrimidine nucleosides end in “-dine”Pyrimidine nucleosides end in “-dine”

Thymidine, Cytidine, UridineThymidine, Cytidine, Uridine

Nucleotides:Nucleotides:– Start with the nucleoside name from above Start with the nucleoside name from above

and add “mono-”, “di-”, or “triphosphate”and add “mono-”, “di-”, or “triphosphate”Adenosine Monophosphate, Cytidine Triphosphate, Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine DiphosphateDeoxythymidine Diphosphate

In-Class ActivitiesIn-Class Activities

Look at the Look at the Nucleotide StructuresNucleotide Structures

Take the Take 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 among “tricks” that help you to distinguish among the different structuresthe different structures

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 bases– First 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 purine) Get broken down into Uric Acid (a purine) Buchanan (mid 1900s) showed where purine Buchanan (mid 1900s) showed where purine ring components came from:ring components came from:

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

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 HOO2-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 + PiFGAM

Synthetase

HC

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole Ribotide (AIR)

ATP

ADP + PiAIR 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

Purine Nucleotide Synthesis Purine Nucleotide Synthesis at a Glanceat a Glance

ATP is involved in 6 stepsATP is involved in 6 steps

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

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

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 2, PPIn step 2, PPii is hydrolyzed to 2P is hydrolyzed to 2P ii (irreversible, “committing” step) (irreversible, “committing” step)

Hydrolyzing a phosphate from ATP is relatively easy Hydrolyzing 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 energy, If endergonic reaction released energy into cell as heat energy, wouldn’t be useful wouldn’t be useful

– Must be coupled to an exergonic reactionMust be coupled to an exergonic reaction

When ATP is a reactant:When ATP is a reactant:

– Part of the ATP can be transferred to an acceptor: PPart of the ATP can be transferred to an acceptor: P ii, PP, PPii, adenyl, , adenyl, or adenosinyl group or adenosinyl group

– ATP hydrolysis can drive an otherwise unfavorable reactionATP hydrolysis can drive an otherwise unfavorable reaction(synthetase; “energase”)(synthetase; “energase”)

Coupling of ReactionsCoupling of Reactions

Purine Biosynthetic PathwayPurine Biosynthetic PathwayChanneling of some reactions on pathway organizes and Channeling of some reactions on pathway organizes and controls processing of substrates to products in each stepcontrols processing of substrates to products in each step– Increases overall rate of pathway and protects intermediates from Increases overall rate of pathway and protects intermediates from

degradationdegradation

In animals, IMP synthesis pathway shows channeling at:In animals, IMP synthesis pathway shows channeling at:– Reactions 3, 4, 6Reactions 3, 4, 6– Reactions 7, 8Reactions 7, 8– Reactions 10, 11Reactions 10, 11

In Class ActivityIn Class Activity******

Calculate how many ATP equivalents are needed for the Calculate how many ATP equivalents are needed for the de novode novo synthesize IMP. synthesize IMP. Assume that all of the substrates (R5P, glutamine, etc) are availableAssume that all of the substrates (R5P, glutamine, etc) are available

Note: You should be able to do this calculation for the synthesis of Note: You should be able to do this calculation for the synthesis of any of the nucleoside monophosphatesany of the nucleoside monophosphates

IMP Conversion to AMP IMP 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 involved in GTP is involved in AMP synthesis and ATP is involved in GMP synthesis (reciprocal control of production)GMP synthesis (reciprocal control of production)PRPP is a biosynthetically “central” molecule (why?)PRPP is a biosynthetically “central” molecule (why?)– ADP/GDP levels – negative feedback on Ribose Phosphate ADP/GDP levels – negative feedback on Ribose Phosphate

Pyrophosphokinase Pyrophosphokinase – Amidophosphoribosyl transferase is activated by PRPP levelsAmidophosphoribosyl transferase is activated by PRPP levels– APRT 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 BiosynthesisRegulatory Control of Purine Biosynthesis

Above the level of IMP production:Above the level of IMP production:– Independent controlIndependent control– Synergistic controlSynergistic control– Feedforward activation by PRPPFeedforward activation by PRPP

Below level of IMP productionBelow level of IMP production– Reciprocal controlReciprocal control

Total amounts of purine nucleotides controlledTotal amounts of purine nucleotides controlledRelative amounts of ATP, GTP controlledRelative amounts of ATP, GTP controlled

Purine Catabolism and SalvagePurine Catabolism and Salvage

All purine degradation leads to All purine degradation leads to uric acid uric acid (but it might not (but it might not stop there)stop there)Ingested nucleic acids are degraded to nucleotides by Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases pancreatic nucleases, and intestinal phosphodiesterases in the intestinein the intestineGroup-specific nucleotidases and non-specific Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosidesphosphatases degrade nucleotides into nucleosides– Direct absorption of nucleosides Direct absorption of nucleosides – Further degradation Further 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 AND NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED.EXCRETED.

Intracellular Purine CatabolismIntracellular Purine CatabolismNucleotides broken into nucleosides by action of Nucleotides broken into nucleosides by action of 5’-nucleotidase (hydrolysis reactions)5’-nucleotidase (hydrolysis reactions)Purine nucleoside phosphorylase (PNP)Purine nucleoside phosphorylase (PNP)– Inosine Inosine Hypoxanthine Hypoxanthine– Xanthosine Xanthosine Xanthine Xanthine– Guanosine Guanosine Guanine Guanine– Ribose-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 CatabolismIntracellular Purine Catabolism

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

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

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

Adenosine 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 Oxidase Xanthine Oxidase

A homodimeric proteinA homodimeric proteinContains electron transfer proteinsContains electron transfer proteins– FADFAD– Mo-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 + P AMP + Fumarate + GDP + Pii (Adenylosuccinate Synthetase)(Adenylosuccinate Synthetase)

COMBINE THE TWO REACTIONS:COMBINE 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 Aspartate to The overall result of combining reactions is deamination of Aspartate to Fumarate at the expense of a GTP Fumarate at the expense of a GTP

THE PURINE NUCLEOTIDE CYCLETHE PURINE NUCLEOTIDE CYCLE

Purine Nucleotide CyclePurine Nucleotide Cycle******

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

Uric Acid ExcretionUric Acid Excretion

Humans – excreted into urine as insoluble Humans – excreted into urine as insoluble crystalscrystalsBirds, terrestrial reptiles, some insects – Birds, terrestrial reptiles, some insects – excrete insoluble crystals in paste form excrete insoluble crystals in paste form – Excess amino N converted to uric acidExcess amino N converted to uric acid

(conserves water)(conserves water)

Others – further modification :Others – further modification :

Uric Acid Uric Acid Allantoin Allantoin Allantoic Acid Allantoic Acid Urea Urea Ammonia Ammonia

Purine Purine SalvageSalvage

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

Hypoxanthine-Guanine phosphoribosyl transferase Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT)(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 resynthesized AMP,IMP,GMP do not need to be resynthesized de novo !de novo !

A CASE STUDY : GOUTA CASE STUDY : GOUTA 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A PAINFUL A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A PAINFUL AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT HE HAD EATEN A MEAL OF FRIED LIVER AND ONIONS, AFTER HE HAD EATEN A MEAL OF FRIED LIVER AND ONIONS, AFTER WHICH HE MET WITH HIS POKER GROUP AND DRANK A WHICH HE MET WITH HIS POKER GROUP AND DRANK A NUMBER OF BEERS.NUMBER OF BEERS.HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS” HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS” WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL < SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL < 7.0 mg/dL).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, OFTEN GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS, OFTEN COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEET.COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEET.

A CASE STUDY : GOUTA CASE STUDY : GOUTTHE DOCTOR RECOMMENDED THAT THE MAN USE THE DOCTOR RECOMMENDED THAT THE MAN USE NSAIDS FOR PAIN AND SWELLING, INCREASE HIS NSAIDS FOR PAIN AND SWELLING, INCREASE HIS FLUID INTAKE (BUT NOT WITH ALCOHOL) AND REST FLUID INTAKE (BUT NOT WITH ALCOHOL) AND REST AND ELEVATE HIS FOOT. HE ALSO PRESCRIBED AND ELEVATE HIS FOOT. HE ALSO PRESCRIBED ALLOPURINOL. 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 THE OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE MAN SOME ADVICE REGARDING LIFE STYLE MAN SOME ADVICE REGARDING LIFE STYLE CHANGES.CHANGES.

GoutGout Impaired excretion or overproduction of uric Impaired excretion or overproduction of uric

acidacid Uric acid crystals precipitate into joints Uric acid crystals precipitate into joints

(Gouty Arthritis), kidneys, ureters (stones)(Gouty Arthritis), kidneys, ureters (stones) Lead impairs uric acid excretion – lead Lead impairs uric acid excretion – lead

poisoning from pewter drinking gobletspoisoning from pewter drinking goblets Fall of Roman Empire?Fall 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 to analog that binds to Xanthine Oxidase to decrease uric acid productiondecrease uric acid production

ALLOPURINOL IS A XANTHINE OXIDASE ALLOPURINOL IS A XANTHINE OXIDASE INHIBITORINHIBITOR

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

Choi HK, Atkinson K, Karlson EW et al. . 2004. “Alcohol intake and risk of incident gout in men:a prospective study”. Lancet 363: 1277-1281

ALCOHOL CONSUMPTION AND GOUT

Lesch-Nyhan SyndromeLesch-Nyhan Syndrome A defect in production or activity ofA defect in production or activity of HGPRT HGPRT

Causes increased level of Hypoxanthine and Causes increased level of Hypoxanthine and Guanine (Guanine ( in degradation to uric acid) in degradation to uric acid)

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

(and thereby increases their degradation)(and thereby increases their degradation) Causes gout-like symptoms, but also Causes gout-like symptoms, but also

neurological symptoms neurological symptoms spasticity, spasticity, aggressiveness, self-mutilationaggressiveness, self-mutilation

First neuropsychiatric abnormality that First neuropsychiatric abnormality that was attributed to a single enzymewas attributed to a single enzyme

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

overproduce purinesoverproduce purines To diagnose, must test urine over To diagnose, must test urine over

24 hours24 hours Biochemical findings from this test Biochemical findings from this test

disappear in adolescencedisappear in adolescence Must obtain urine specimen in Must obtain urine specimen in

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

be seen in diapersbe seen in diapers

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

• EXPLAIN THE BIOCHEMICAL EVENTS THAT LEAD EXPLAIN THE BIOCHEMICAL EVENTS THAT LEAD TO INCREASED URIC ACID PRODUCTION?TO INCREASED URIC ACID PRODUCTION?

• WHY DOES HYPOGLYCEMIA OCCUR IN THIS WHY DOES HYPOGLYCEMIA OCCUR IN THIS DISEASE?DISEASE?

• WHY IS THE LIVER ENLARGED?WHY IS THE LIVER ENLARGED?

Pyrimidine Ribonucleotide Pyrimidine Ribonucleotide SynthesisSynthesis

Uridine Monophosphate (UMP) is Uridine Monophosphate (UMP) is synthesized firstsynthesized first• CTP is synthesized from UMPCTP is synthesized from UMP

Pyrimidine 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

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 HOO2-O3P

Orotidine-5'-monophosphate(OMP)

HN

CN

CH

CHC

O

O

HH

CH2

OH OH

H HOO2-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 Synthesis OverviewUMP Synthesis Overview 2 ATPs needed: both used in first step2 ATPs needed: both used in first step

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

2 condensation rxns: form carbamoyl aspartate 2 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 reduction

Attachment 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-P• PPPPii 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

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

FINAL REACTION OF PYRIMIDINE PATHWAYFINAL REACTION OF PYRIMIDINE PATHWAY ANOTHER MECHANISM FOR DECARBOXYLATIONANOTHER MECHANISM FOR DECARBOXYLATION A HIGH ENERGY CARBANION INTERMEDIATE NOT A HIGH ENERGY CARBANION INTERMEDIATE NOT

NEEDEDNEEDED NO COFACTORS NEEDED !NO COFACTORS NEEDED ! SOME OF THE BINDING ENERGY BETWEEN OMP SOME OF THE BINDING ENERGY BETWEEN OMP

AND THE ACTIVE SITE IS USED TO STABILIZE THE AND THE ACTIVE SITE IS USED TO STABILIZE THE TRANSITION STATETRANSITION STATE• ““PREFERENTIAL TRANSITION STATE BINDING”PREFERENTIAL TRANSITION STATE BINDING”

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 UDP; to UMP to form UDP; nucleoside diphosphate kinase catalyzes nucleoside diphosphate kinase catalyzes transfer of Ptransfer of Pii from ATP to UDP to form UTP from ATP to UDP to form UTP

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

• Glutamine provides amide nitrogen for CGlutamine provides amide nitrogen for C4 4 in animalsin animals

Regulatory Control of Pyrimidine Regulatory Control of Pyrimidine SynthesisSynthesis

Differs between bacteria and animalsDiffers between bacteria and animals• Bacteria – 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 itUDP and UTP inhibit enzyme; ATP and PRPP activate it• 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 Aciduria Caused 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 urine urine

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 (all others would be lethal to fetus)to fetus)

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

CYTIDINE ADMINISTRATION WORK TO TREAT CYTIDINE ADMINISTRATION WORK TO TREAT OROTIC ACIDURIA?OROTIC ACIDURIA?

Degradation of PyrimidinesDegradation of Pyrimidines CMP and UMP degraded to bases CMP and UMP degraded to bases

similarly to purines similarly to purines • DephosphorylationDephosphorylation• DeaminationDeamination• Glycosidic bond cleavageGlycosidic bond cleavage

Uracil reduced in liver, forming Uracil reduced in liver, forming --alanine alanine • Converted to malonyl-CoA Converted to malonyl-CoA fatty acid fatty acid

synthesis for energy metabolismsynthesis for energy metabolism

Deoxyribonucleotide FormationDeoxyribonucleotide Formation 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 ribonucleotide production ribonucleotide production

Deoxyribonucleotides are synthesized Deoxyribonucleotides are synthesized from corresponding ribonucleotides from 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 DNA• Thymine (5-methyluracil) appears insteadThymine (5-methyluracil) appears instead

Formation of DeoxyribonucleotidesFormation of Deoxyribonucleotides

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 SUBUNIT• Three allosteric sitesThree allosteric sites

Specificity SiteSpecificity Site Hexamerization siteHexamerization site Activity SiteActivity Site

• Five redox-active –SH groups from cysteinesFive redox-active –SH groups from cysteines

R2 SUBUNITR2 SUBUNIT• Tyr 122 radicalTyr 122 radical• Binuclear Fe(III) complexBinuclear Fe(III) complex

Ribonucleotide Reductase R2 Ribonucleotide Reductase R2 Subunit 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 provides the reducing equivalentsgroups provides the reducing equivalents• A protein disulfide group is formedA protein disulfide group is formed• Gets reduced by two other sulfhydryl gps of Gets reduced by two other sulfhydryl gps of

Cys residues in R1Cys residues in R1

Chime ExerciseE. coli Ribonucleotide Reductase:

3R1R and 4R1R: R1 subunit1RIB and 1AV8: R2 subunit

 • Explore 1AV8: Ribonucleotide Reductase in detail.This is the R2

subunit of E. coli Ribonucleotide Reductase.  The biological molecule consists of a heterotetramer of 2 R1 and two R2 chains.

 • Identify the following structures: 

– 8 long -helices in one unit of R2– Tyr 122 residue– The binuclear Fe (III) complex– The ligands of the Fe (III) complex 

Mechanism of Ribonucleotide Reductase Mechanism of Ribonucleotide Reductase ReactionReaction

Free RadicalFree Radical Involvement of multiple –SH groupsInvolvement of multiple –SH groups RR 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 CELLULAR ACTIVITY IS RESPONSIVE TO LEVEL OF CELLULAR NUCLEOTIDES:NUCLEOTIDES:• ATP ACTIVATES REDUCTION OFATP ACTIVATES REDUCTION OF

CDPCDP UDPUDP

• dTTP dTTP INDUCES GDP REDUCTIONINDUCES GDP REDUCTION INHIBITS REDUCTION OF CDP. UDPINHIBITS REDUCTION OF CDP. UDP

• dATP INHIBITS REDUCTION OF ALL NUCLEOTIDESdATP INHIBITS REDUCTION OF ALL NUCLEOTIDES• dGTP dGTP

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

RIBONUCLEOTIDE REDUCTASERIBONUCLEOTIDE REDUCTASE

CATALYTIC ACTIVITY VARIES WITH STATE OF CATALYTIC ACTIVITY VARIES WITH STATE OF OLIGOMERIZATION:OLIGOMERIZATION:• WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE

OF R1 (CATALYTICALLY INACTIVE MONOMER)OF R1 (CATALYTICALLY INACTIVE MONOMER) CATALYTICALLY ACTIVE (R1)CATALYTICALLY ACTIVE (R1)22

• WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERSWHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERS TETRAMER FORMATIONTETRAMER FORMATION (R1)(R1)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

ThioredoxinThioredoxin Physiologic reducing agent of RNRPhysiologic reducing agent of RNR Cys pair can swap H atoms with disulfide Cys pair can swap H atoms with disulfide

formed formed regenerate original enzymeregenerate original enzyme• Thioredoxin gets oxidized to disulfideThioredoxin gets oxidized to disulfide

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

Thymine FormationThymine Formation Formed by methylating deoxyuridine Formed by methylating deoxyuridine

monophosphate (dUMP) monophosphate (dUMP) UTP is needed for RNA production, but UTP is 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 dUTPase dUTP hydrolyzed by dUTPase (dUTP diphosphohydrolase) to dUMP (dUTP diphosphohydrolase) to dUMP

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

CHIME EXERCISE: dUTPaseCHIME EXERCISE: dUTPase 1DUD: Deoxyuridine-5'-Nucleotide Hydrolase in a 1DUD: Deoxyuridine-5'-Nucleotide Hydrolase in a

complex with a bound substrate analog, complex with a bound substrate analog, Deoxyuridine-5'-Diphosphate (dUDP). Deoxyuridine-5'-Diphosphate (dUDP).

Explore dUTPase as follows:Explore dUTPase as follows:

• Find the substrate in its binding siteFind the substrate in its binding site• Find C5 on the Uracil group. Is there enough room to Find C5 on the Uracil group. Is there enough room to

attach a methyl group to C5?attach a methyl group to C5?• Locate the ribose 2’ C. What protein group sterically Locate the ribose 2’ C. What protein group sterically

prevents an –OH group from being attached to the 2’ C prevents an –OH group from being attached to the 2’ C atom?atom?

• Find the H-bond donors and acceptors (to the uracil Find the H-bond donors and acceptors (to the uracil base) from the protein. What would be the effect on the base) from the protein. What would be the effect on the H-bonding if the base was changed to cytosine?H-bonding if the base was changed to cytosine?

Tetrahydrofolate (THF)Tetrahydrofolate (THF) Methylation of dUMP catalyzed by Methylation of dUMP catalyzed by

thymidylate synthase thymidylate synthase • Cofactor: NCofactor: N55,N,N1010-methylene THF-methylene THF

Oxidized to dihydrofolateOxidized to dihydrofolate Only known rxn where net oxidation state Only known rxn where net oxidation state

of THF changesof THF changes THF Regeneration:THF 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 transferase)(enzyme: serine hydroxymethyl transferase)

dUMP dTMP

NADPH + H+

NADP+

SERINE

GLYCINE

REGENERATION OF N5,N10 METHYLENETETRAHYDROFOLATE

DHFN5,N10 – METHYLENE-THF

THF

dihydrofolate reductaseserine hydroxymethyl transferase

thymidylate synthase

dUMP dTMP

NADPH + H+

NADP+

SERINE

GLYCINE

INHIBITORS OF N5,N10 METHYLENETETRAHYDROFOLATE REGENERATION

DHFN5,N10 – METHYLENE-THF

THF

dihydrofolate reductaseserine hydroxymethyl transferase

thymidylate synthase

METHOTREXATE AMINOPTERIN TRIMETHOPRIM

FdUMP

X

X

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 production decrease dTMP production (fluorodeoxyuridylate – irreversible inhibitor) – also (fluorodeoxyuridylate – irreversible inhibitor) – 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

ADENOSINE DEAMINASE DEFICIENCYADENOSINE DEAMINASE DEFICIENCY

IN PURINE DEGRADATION, ADENOSINE IN PURINE DEGRADATION, ADENOSINE INOSINE INOSINE• ENZYME IS ADAENZYME IS ADA

ADA DEFICIENCY RESULTS IN SCIDADA DEFICIENCY RESULTS IN SCID• ““SEVERE COMBINED IMMUNODEFICIENCY”SEVERE COMBINED IMMUNODEFICIENCY”

SELECTIVELY KILLS LYMPHOCYTESSELECTIVELY KILLS LYMPHOCYTES• BOTH B- AND T-CELLSBOTH B- AND T-CELLS• MEDIATE MUCH OF IMMUNE RESPONSEMEDIATE MUCH OF IMMUNE RESPONSE

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

Adenosine DeaminaseAdenosine DeaminaseCHIME Exercise: 2ADACHIME Exercise: 2ADA

Enzyme catalyzing deamination of Adenosine to Inosine Enzyme catalyzing deamination of Adenosine to Inosine // barrel domain structure barrel domain structure– ““TIM Barrel” – central barrel structure with 8 twisted TIM Barrel” – central barrel structure with 8 twisted

parallel parallel -strands connected by 8 -strands connected by 8 -helical loops-helical loops– Active site is at bottom of funnel-shaped pocket Active site is at bottom of funnel-shaped pocket

formed by loopsformed by loops– Found in all glycolytic enzymesFound in all glycolytic enzymes– Found in proteins that bind and transport metabolitesFound in proteins that bind and transport metabolites

ADA DEFICIENCYADA DEFICIENCY******

IN-CLASS QUESTION: EXPLAIN THE BIOCHEMISTRY IN-CLASS QUESTION: EXPLAIN THE BIOCHEMISTRY THAT RESULTS WHEN A PERSON HAS ADA THAT RESULTS WHEN A PERSON HAS ADA DEFICIENCYDEFICIENCY

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

ADA DEFICIENCYADA DEFICIENCY ONE OF FIRST DISEASES TO BE TREATED WITH ONE OF FIRST DISEASES TO BE TREATED WITH

GENE THERAPYGENE THERAPY

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

PEG-ADA TREATMENTSPEG-ADA TREATMENTS• ACTIVITY LASTS 1-2 WEEKSACTIVITY LASTS 1-2 WEEKS