Nucleotides: Synthesis and

Degradation

Nitrogenous Bases

Planar, aromatic, and heterocyclic

Derived from purine or pyrimidine

Numbering of bases is “unprimed”

Nucleic Acid Bases

Purines Pyrimidines

Sugars

Pentoses (5-C sugars)

Numbering of sugars is “primed”

Sugars

D-Ribose and 2’-Deoxyribose

*Lacks a 2’-OH group

Nucleosides

Result from linking one of the sugars with

a purine or pyrimidine base through an N-

glycosidic linkage

– Purines bond to the C1’ carbon of the sugar at

their N9 atoms

– Pyrimidines bond to the C1’ carbon of the

sugar at their N1 atoms

Nucleosides

Phosphate Groups

Mono-, di- or triphosphates

Phosphates can be bonded to either C3 or

C5 atoms of the sugar

Nucleotides

Result from linking one or more phosphates

with a nucleoside onto the 5’ end of the

molecule through esterification

Nucleotides

RNA (ribonucleic acid) is a polymer of

ribonucleotides

DNA (deoxyribonucleic acid) is a polymer

of deoxyribonucleotides

Both deoxy- and ribonucleotides contain

Adenine, Guanine and Cytosine

– Ribonucleotides contain Uracil

– Deoxyribonucleotides contain Thymine

Nucleotides

Monomers for nucleic acid polymers

Nucleoside Triphosphates are important

energy carriers (ATP, GTP)

Important components of coenzymes

– FAD, NAD+ and Coenzyme A

Naming Conventions

Nucleosides:

– Purine nucleosides end in “-sine”

Adenosine, Guanosine

– Pyrimidine nucleosides end in “-dine”Thymidine, Cytidine, Uridine

Nucleotides:

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

Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate

In-Class Activities

Look at the Nucleotide Structures

Take the Nucleotide Identification Quiz

Be prepared to identify some of these

structures on an exam. Learn some

“tricks” that help you to distinguish among

the different structures

Nucleotide MetabolismPURINE RIBONUCLEOTIDES: formed de novo– i.e., purines are not initially synthesized as free bases

– First purine derivative formed is Inosine Mono-phosphate (IMP)

The purine base is hypoxanthine

AMP and GMP are formed from IMP

Purine Nucleotides

Get broken down into Uric Acid (a purine)

Buchanan (mid 1900s) showed where purine

ring components came from:

N1: Aspartate AmineC2, C8: Formate

N3, N9: GlutamineC4, C5, N7: Glycine

C6: Bicarbonate Ion

Purine Nucleotide Synthesis

OH

H

H

CH2

OH OH

H HO

α

O2-O3P

α-D-Ribose-5-Phosphate (R5P)

O

H

H

CH2

OH OH

H HO α

O2-O3P

5-Phosphoribosyl-α-pyrophosphate (PRPP)

P

O

O

O P

O

O

O

ATP

AMP

RibosePhosphatePyrophosphokinase

H

NH2

H

CH2

OH OH

H HO

β

O2-O3P

β-5-Phosphoribosylamine (PRA)

Amido phosphoribosyl

Transf erase

Glutamine + H2O

Glutamate

+ PPi

H

NH

H

CH2

OH OH

H HO

O2-O3P

CO

H2C NH2

Glycinamide Riboti de (GAR)

GAR Syn thetase

Glycine + ATP

ADP

+ Pi

H2C

C

NH

O

CH

HN

O

Ribose-5-Phosphate

Formylglycinamide ribotide (FGAR)

H2C

C

NH

O

CH

HN

HN

Ribose-5-Phosphate

Formylglycinamidine ribotide (FGAM)

THFN10-Formyl-THF

GAR Transformylase

ATP +

Glutamine +

H 2O

ADP +

Glutamate + PiFGAM

Synthetase

HC

CN

CH

N

H2N

Ribose-5-Phosphate

4

5

5-Aminoimidazole Ribo tide (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

+ ATPADP

+ Pi

SAICAR Synthetase

Adenylosuccinate

Lyase

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-carboxamide

ribotide (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

HC

N

C

C

C

N

CH

N

O

4

5

HH

CH2

OH OH

H HO

O2-O3P

IMP

Cyclohydrolase

H2O

Purine Nucleotide Synthesis at a Glance

ATP is involved in 6 steps

PRPP in the first step of Purine synthesis is also a precursor for

Pyrimidine Synthesis, His and Trp synthesis

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

coupling

In second step, C1 notation changes from α to β (anomers specifying OH positioning on C1 with respect to C4 group)

In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)

Hydrolyzing a phosphate from ATP is relatively easy

∆G°’= -30.5 kJ/mol

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

– Must be coupled to an exergonic reaction

When ATP is a reactant:

– Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl, or adenosinyl group

– ATP hydrolysis can drive an otherwise unfavorable reaction

(synthetase; “energase”)

Coupling of Reactions

Purine Biosynthetic Pathway

Channeling of some reactions on pathway organizes and

controls processing of substrates to products in each step

– Increases overall rate of pathway and protects intermediates from

degradation

In animals, IMP synthesis pathway shows channeling at:

– Reactions 3, 4, 6

– Reactions 7, 8

– Reactions 10, 11

In Class Activity

***

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

Note: You should be able to do this calculation for the synthesis of

any of the nucleoside monophosphates

IMP Conversion to AMP

IMP Conversion to GMP

Regulatory Control of Purine

Nucleotide BiosynthesisGTP is involved in AMP synthesis and ATP is involved

in GMP synthesis (reciprocal control of production)

PRPP is a biosynthetically “central” molecule (why?)

– ADP/GDP levels – negative feedback on Ribose Phosphate

Pyrophosphokinase

– Amidophosphoribosyl transferase is activated by PRPP levels

– APRT activity has negative feedback at two sites

ATP, ADP, AMP bound at one site

GTP,GDP AND GMP bound at the other site

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

increases with increasing concentrations of ATP

Regulatory Control of Purine Biosynthesis

Above the level of IMP production:

– Independent control

– Synergistic control

– Feedforward activation by PRPP

Below level of IMP production

– Reciprocal control

Total amounts of purine nucleotides controlled

Relative amounts of ATP, GTP controlled

Purine Catabolism and Salvage

All purine degradation leads to uric acid (but it might not stop there)Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestineGroup-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides– Direct absorption of nucleosides

– Further degradation

Nucleoside + H2O � base + ribose (nucleosidase)

Nucleoside + Pi � base + r-1-phosphate (n. phosphorylase)

NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED.

Intracellular Purine Catabolism

Nucleotides broken into nucleosides by action of

5’-nucleotidase (hydrolysis reactions)

Purine nucleoside phosphorylase (PNP)

– Inosine � Hypoxanthine

– Xanthosine � Xanthine

– Guanosine � Guanine

– Ribose-1-phosphate splits off

Can be isomerized to ribose-5-phosphate

Adenosine is deaminated to Inosine (ADA)

Intracellular Purine Catabolism

Xanthine is the point of convergence for the

metabolism of the purine bases

Xanthine � Uric acid

– Xanthine oxidase catalyzes two reactions

Purine ribonucleotide degradation pathway

is same for purine deoxyribonucleotides

Adenosine Degradation

Xanthosine 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

A homodimeric protein

Contains electron transfer proteins

– FAD

– Mo-pterin complex in +4 or +6 state

– Two 2Fe-2S clusters

Transfers electrons to O2 � H2O2

– H2O2 is toxic

– Disproportionated to H2O and O2 by catalase

AMP + H2O � IMP + NH4+ (AMP Deaminase)

IMP + Aspartate + GTP � AMP + Fumarate + GDP + Pi(Adenylosuccinate Synthetase)

COMBINE THE TWO REACTIONS:

Aspartate + H2O + GTP � Fumarate + GDP + Pi + NH4+

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

THE PURINE NUCLEOTIDE CYCLE

Purine Nucleotide Cycle

***

In-Class Question: Why is the purine nucleotide

cycle important in muscle metabolism during a

burst of activity?

Uric Acid Excretion

Humans – excreted into urine as insoluble

crystals

Birds, terrestrial reptiles, some insects –

excrete insoluble crystals in paste form

– Excess amino N converted to uric acid

(conserves water)

Others – further modification :

Uric Acid � Allantoin � Allantoic Acid � Urea � Ammonia

Purine Salvage

Adenine phosphoribosyl transferase (APRT)

Adenine + PRPP � AMP + PPi

Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT)

Hypoxanthine + PRPP � IMP + PPi

Guanine + PRPP � GMP + PPi

(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)

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

A CASE STUDY : GOUT

A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A PAINFUL

AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT

HE HAD EATEN A MEAL OF FRIED LIVER AND ONIONS, AFTER

WHICH HE MET WITH HIS POKER GROUP AND DRANK A

NUMBER OF BEERS.

HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS”

WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS

SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL <

7.0 mg/dL).

THE MAN RECALLED THAT HIS FATHER AND HIS

GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS, OFTEN

COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEET.

A CASE STUDY : GOUT

THE DOCTOR RECOMMENDED THAT THE MAN USE

NSAIDS FOR PAIN AND SWELLING, INCREASE HIS

FLUID INTAKE (BUT NOT WITH ALCOHOL) AND

REST AND ELEVATE HIS FOOT. HE ALSO

PRESCRIBED ALLOPURINOL.

A FEW DAYS LATER THE CONDITION HAD

RESOLVED AND ALLOPURINOL HAD BEEN

STOPPED. A REPEAT URIC ACID LEVEL WAS

OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE MAN SOME ADVICE REGARDING LIFE STYLE

CHANGES.

Gout

� Impaired excretion or overproduction of uric acid

� Uric acid crystals precipitate into joints (Gouty Arthritis), kidneys, ureters (stones)

� Lead impairs uric acid excretion – lead poisoning from pewter drinking goblets� Fall of Roman Empire?

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

� Allopurinol treatment – hypoxanthine analog that binds to Xanthine Oxidase to decrease uric acid production

ALLOPURINOL IS A XANTHINE OXIDASE

INHIBITOR

A SUBSTRATE ANALOG IS CONVERTED TO AN

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 Syndrome

� A defect in production or activity of

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

� Also,PRPP accumulates

� stimulates production of purine nucleotides (and thereby increases their degradation)

� Causes gout-like symptoms, but also neurological symptoms � spasticity, aggressiveness, self-mutilation

� First neuropsychiatric abnormality that was attributed to a single enzyme

Purine Autism

� 25% of autistic patients may

overproduce purines

� To diagnose, must test urine over

24 hours

� Biochemical findings from this test

disappear in adolescence

� Must obtain urine specimen in

infancy, but it’s difficult to do!

• Pink urine due to uric acid crystals may

be seen in diapers

IN-CLASS QUESTION

***

� IN von GIERKE’S DISEASE, OVERPRO-DUCTION OF URIC ACID OCCURS. THIS DISEASE IS CAUSED BY A DEFICIENCY OF GLUCOSE-6-PHOSPHATASE.

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

• WHY DOES HYPOGLYCEMIA OCCUR IN THIS DISEASE?

• WHY IS THE LIVER ENLARGED?

Pyrimidine Ribonucleotide

Synthesis

� Uridine Monophosphate (UMP) is synthesized first

• CTP is synthesized from UMP

� Pyrimidine ring synthesis completed first; then attached to ribose-5-phosphate

N1, C4, C5, C6 : Aspartate

C2 : HCO3-

N3 : Glutamine amide Nitrogen

2 ATP + HCO3- + Glutamine + H2O

CO

O PO3-2

NH2

Carbamoyl Phosphate

NH2

C

N

H

CH

CH2

C

COOO

HO

O

Carbamoyl Aspartate

HN

C

N

H

CH

CH2

C

COOO

O

Dihydroorotate

HN

C

N

H

C

CH

C

COOO

O

Orotate

HN

C

N

C

CH

C

COOO

O

HH

CH2

OH OH

H H

OO2-O3P

β

Orotidine-5'-monophosphate

(OMP)

HN

C

N

CH

CH

C

O

O

HH

CH2

OH OH

H H

OO

2-O3P

β

Uridine Monophosphate

(UMP)

2 ADP +

Glutamate +

Pi

CarbamoylPhosphateSynthetase II

Aspartate

Transcarbamoylase

(ATCase)

Aspartate

Pi

H2O

Dihydroorotase

Quinone

Reduced

QuinoneDihydroorotateDehydrogenase

PRPP PPi

Orotate PhosphoribosylTransferase

CO2

OMP

Decarboxylase

Pyrimidine Synthesis

UMP Synthesis Overview� 2 ATPs needed: both used in first step

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

� 2 condensation rxns: form carbamoyl aspartate and dihydroorotate (intramolecular)

� Dihydroorotate dehydrogenase is an intra-mitochondrial enzyme; oxidizing power comes from quinone reduction

� Attachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-P• PPi splits off PRPP – irreversible

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

OMP DECARBOXYLASE : THE MOST

CATALYTICALLY PROFICIENT ENZYME

� FINAL REACTION OF PYRIMIDINE PATHWAY

� ANOTHER MECHANISM FOR DECARBOXYLATION

� A HIGH ENERGY CARBANION INTERMEDIATE NOT NEEDED

� NO COFACTORS NEEDED !

� SOME OF THE BINDING ENERGY BETWEEN OMP AND THE ACTIVE SITE IS USED TO STABILIZE THE TRANSITION STATE

• “PREFERENTIAL TRANSITION STATE BINDING”

UMP � UTP and CTP

� Nucleoside monophosphate kinase catalyzes transfer of Pi to UMP to form

UDP; nucleoside diphosphate kinase

catalyzes transfer of Pi from ATP to UDP to form UTP

� CTP formed from UTP via CTP Synthetasedriven by ATP hydrolysis

• Glutamine provides amide nitrogen for C4 in animals

Regulatory Control of Pyrimidine

Synthesis

� Differs between bacteria and animals

• Bacteria – regulation at ATCase rxn

� Animals – regulation at carbamoyl phosphate synthetase II

• UDP and UTP inhibit enzyme; ATP and PRPP activate it

• UMP and CMP competitively inhibit OMP Decarboxylase

*Purine synthesis inhibited by ADP and GDP at

ribose phosphate pyrophosphokinase step, controlling level of PRPP � also regulates

pyrimidines

Orotic Aciduria

� Caused by defect in protein chain with enzyme activities of last two steps of pyrimidine synthesis

� Increased excretion of orotic acid in urine

� Symptoms: retarded growth; severe anemia

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

� Treat with uridine/cytidine� IN-CLASS QUESTION: HOW DOES URIDINE AND CYTIDINE ADMINISTRATION WORK TO TREAT OROTIC ACIDURIA?

Degradation of Pyrimidines

� CMP and UMP degraded to bases similarly to purines • Dephosphorylation

• Deamination

• Glycosidic bond cleavage

� Uracil reduced in liver, forming β-alanine • Converted to malonyl-CoA � fatty acid synthesis for energy metabolism

Deoxyribonucleotide Formation

� Purine/Pyrimidine degradation are the same for ribonucleotides and

deoxyribonucleotides

� Biosynthetic pathways are only for ribonucleotide production

� Deoxyribonucleotides are synthesized from corresponding ribonucleotides

DNA vs. RNA: REVIEW

� DNA composed of deoxyribonucleotides

� Ribose sugar in DNA lacks hydroxyl group

at 2’ Carbon

� Uracil doesn’t (normally) appear in DNA

• Thymine (5-methyluracil) appears instead

Formation of Deoxyribonucleotides

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

“Ribonucleotide Reductases”

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

� Two subunits: R1 and R2

• A Heterotetramer: (R1)2 and (R2)2 in vitro

RIBONUCLEOTIDE REDUCTASE

� R1 SUBUNIT• Three allosteric sites

� Specificity Site

� Hexamerization site

� Activity Site

• Five redox-active –SH groups from cysteines

� R2 SUBUNIT• Tyr 122 radical

• Binuclear Fe(III) complex

Ribonucleotide Reductase R2

Subunit

� Fe prosthetic group– binuclear, with each Fe octahedrally coordinated • Fe’s are bridged by O-2 and carboxyl gp of Glu 115

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

� During the overall process, a pair of –SH groups provides the reducing equivalents• A protein disulfide group is formed• Gets reduced by two other sulfhydryl gps of Cys residues in R1

Chime Exercise

E. 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 αααα -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

Reaction

� Free Radical

� Involvement of multiple –SH groups

� RR is left with a disulfide group that must be reduced to return to the

original enzyme

RIBONUCLEOTIDE REDUCTASE

� ACTIVITY IS RESPONSIVE TO LEVEL OF CELLULAR NUCLEOTIDES:• ATP ACTIVATES REDUCTION OF

� CDP

� UDP

• dTTP

� INDUCES GDP REDUCTION

� INHIBITS REDUCTION OF CDP. UDP

• dATP INHIBITS REDUCTION OF ALL NUCLEOTIDES

• dGTP

� STIMULATES ADP REDUCTION

� INHIBITS CDP,UDP,GDP REDUCTION

RIBONUCLEOTIDE REDUCTASE

� CATALYTIC ACTIVITY VARIES WITH STATE OF OLIGOMERIZATION:• WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE OF R1 (CATALYTICALLY INACTIVE MONOMER)

� � CATALYTICALLY ACTIVE (R1)2

• WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERS

� � TETRAMER FORMATION

� (R1)4a (ACTIVE STATE) == (R1)4b (INACTIVE)

• WHEN ATP BINDS TO HEXAMERIZATION SITE

� � CATALYTICALLY ACTIVE HEXAMERS (R1)6

Thioredoxin� Physiologic reducing agent of RNR

� Cys pair can swap H atoms with disulfide formed �regenerate original enzyme

• Thioredoxin gets oxidized to disulfide

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

Thymine Formation

� Formed by methylating deoxyuridine monophosphate (dUMP)

� UTP is needed for RNA production, but dUTP not needed for DNA• If dUTP produced excessively, would cause substitution errors (dUTP for dTTP)

� dUTP hydrolyzed by dUTPase

(dUTP diphosphohydrolase) to dUMP �methylated at C5 to form dTMP�rephosphorylate to form dTTP

CHIME EXERCISE: dUTPase

� 1DUD: Deoxyuridine-5'-Nucleotide Hydrolase in a complex with a bound substrate analog, Deoxyuridine-5'-Diphosphate (dUDP).

� Explore dUTPase as follows:

• Find the substrate in its binding site

• Find C5 on the Uracil group. Is there enough room to attach a methyl group to C5?

• Locate the ribose 2’ C. What protein group sterically prevents an –OH group from being attached to the 2’ C atom?

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

Tetrahydrofolate (THF)

� Methylation of dUMP catalyzed by thymidylate synthase • Cofactor: N5,N10-methylene THF

� Oxidized to dihydrofolate

� Only known rxn where net oxidation state of THF changes

� THF Regeneration:

DHF + NADPH + H+ � THF + NADP+ (enzyme: dihydrofolate

reductase)

THF + Serine � N5,N10-methylene-THF + Glycine

(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

METHOTREXATEAMINOPTERINTRIMETHOPRIM

FdUMP

X

X

Anti-Folate Drugs

� Cancer cells consume dTMP quickly for DNA replication

• Interfere with thymidylate synthase rxn to decrease dTMP production

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

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

• Anti-Folates: Aminopterin, methotrexate, trimethoprim

ADENOSINE DEAMINASE DEFICIENCY

� IN PURINE DEGRADATION, ADENOSINE �

INOSINE• ENZYME IS ADA

� ADA DEFICIENCY RESULTS IN SCID

• “SEVERE COMBINED IMMUNODEFICIENCY”

� SELECTIVELY KILLS LYMPHOCYTES

• BOTH B- AND T-CELLS

• MEDIATE MUCH OF IMMUNE RESPONSE

� ALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB ACTIVE SITE

Adenosine Deaminase

CHIME Exercise: 2ADA

Enzyme catalyzing deamination of Adenosine to Inosine

α/β barrel domain structure

– “TIM Barrel” – central barrel structure with 8 twisted

parallel β-strands connected by 8 α-helical loops

– Active site is at bottom of funnel-shaped pocket

formed by loops

– Found in all glycolytic enzymes

– Found in proteins that bind and transport metabolites

ADA DEFICIENCY

***

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

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

ADA DEFICIENCY

� ONE OF FIRST DISEASES TO BE TREATED WITH GENE THERAPY

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

� PEG-ADA TREATMENTS• ACTIVITY LASTS 1-2 WEEKS