Nucleotides: Synthesis and Degradation

Nucleotides: Synthesis and Degradation

Nitrogenous BasesPlanar, aromatic, and heterocyclicDerived from purine or pyrimidineNumbering of bases is “unprimed”

Nucleic Acid BasesPurines Pyrimidines

SugarsPentoses (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

NucleotidesResult 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 NucleotidesGet broken down into Uric Acid (a purine)

Buchanan (mid 1900s) showed where purine 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 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

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 to (anomers specifying OH positioning on C1 with respect to C4 group)

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

Coupling of Reactions

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”)

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 Biosynthesis

GTP 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 intestine

Group-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

THE PURINE NUCLEOTIDE CYCLEAMP + 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

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 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.

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 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 : 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 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 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 Synthetase driven by ATP hydrolysis Glutamine provides amide nitrogen for C4 in

animals

Regulatory Control of Pyrimidine SynthesisDiffers between bacteria and animals

Bacteria – regulation at ATCase rxnAnimals – 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 -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 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 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

ThioredoxinPhysiologic reducing agent of RNRCys 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

METHOTREXATE AMINOPTERIN TRIMETHOPRIM

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 DeaminaseCHIME Exercise: 2ADA Enzyme catalyzing deamination of Adenosine to

Inosine / barrel domain structure

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