Nucleotides: Synthesis and Degradation

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

Nucleic Acid BasesPurinesPyrimidines

SugarsPentoses (5-C sugars)Numbering of sugars is primed

Sugars D-Ribose and 2-Deoxyribose*Lacks a 2-OH group

NucleosidesResult 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 atomsPyrimidines bond to the C1 carbon of the sugar at their N1 atoms

Nucleosides

Phosphate GroupsMono-, 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

NucleotidesRNA (ribonucleic acid) is a polymer of ribonucleotidesDNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotidesBoth deoxy- and ribonucleotides contain Adenine, Guanine and CytosineRibonucleotides contain UracilDeoxyribonucleotides contain Thymine

NucleotidesMonomers for nucleic acid polymersNucleoside Triphosphates are important energy carriers (ATP, GTP)Important components of coenzymesFAD, NAD+ and Coenzyme A

Naming ConventionsNucleosides:Purine nucleosides end in -sine Adenosine, GuanosinePyrimidine nucleosides end in -dineThymidine, Cytidine, UridineNucleotides:Start with the nucleoside name from above and add mono-, di-, or triphosphateAdenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate

In-Class ActivitiesLook 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 novoi.e., purines are not initially synthesized as free basesFirst purine derivative formed is Inosine Mono-phosphate (IMP)The purine base is hypoxanthineAMP 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

Purine Nucleotide Synthesis at a GlanceATP 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 a to b (anomers specifying OH positioning on C1 with respect to C4 group)In step 2, PPi is hydrolyzed to 2Pi (irreversible, committing step)

Coupling of ReactionsHydrolyzing a phosphate from ATP is relatively easy G= -30.5 kJ/molIf endergonic reaction released energy into cell as heat energy, wouldnt be useful Must be coupled to an exergonic reactionWhen 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 PathwayChanneling of some reactions on pathway organizes and controls processing of substrates to products in each stepIncreases overall rate of pathway and protects intermediates from degradationIn animals, IMP synthesis pathway shows channeling at:Reactions 3, 4, 6Reactions 7, 8Reactions 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 levelsAPRT activity has negative feedback at two sitesATP, ADP, AMP bound at one siteGTP,GDP AND GMP bound at the other siteRate of AMP production increases with increasing concentrations of GTP; rate of GMP production increases with increasing concentrations of ATP

Regulatory Control of Purine BiosynthesisAbove the level of IMP production:Independent controlSynergistic controlFeedforward activation by PRPPBelow level of IMP productionReciprocal control

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

Purine Catabolism and SalvageAll 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 nucleosidesDirect 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 CatabolismNucleotides broken into nucleosides by action of 5-nucleotidase (hydrolysis reactions)Purine nucleoside phosphorylase (PNP)Inosine HypoxanthineXanthosine XanthineGuanosine GuanineRibose-1-phosphate splits offCan be isomerized to ribose-5-phosphateAdenosine is deaminated to Inosine (ADA)

Intracellular Purine CatabolismXanthine is the point of convergence for the metabolism of the purine bases

Xanthine Uric acidXanthine 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 proteinContains electron transfer proteins FADMo-pterin complex in +4 or +6 state Two 2Fe-2S clustersTransfers 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 Cycle*** In-Class Question: Why is the purine nucleotide cycle important in muscle metabolism during a burst of activity?

Uric Acid ExcretionHumans excreted into urine as insoluble crystalsBirds, 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 SalvageAdenine phosphoribosyl transferase (APRT)Adenine + PRPP AMP + PPi

Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT)Hypoxanthine + PRPP IMP + PPiGuanine + PRPP GMP + PPi

(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)

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

A CASE STUDY : GOUTA 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 : GOUTTHE 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.

GoutImpaired excretion or overproduction of uric acidUric acid crystals precipitate into joints (Gouty Arthritis), kidneys, ureters (stones)Lead impairs uric acid excretion lead poisoning from pewter drinking gobletsFall of Roman Empire?Xanthine oxidase inhibitors inhibit production of uric acid, and treat goutAllopurinol 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: 1277-1281ALCOHOL CONSUMPTION AND GOUT

Lesch-Nyhan SyndromeA 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-mutilationFirst neuropsychiatric abnormality that was attributed to a single enzyme

Purine Autism25% of autistic patients may overproduce purinesTo diagnose, must test urine over 24 hoursBiochemical findings from this test disappear in adolescenceMust obtain urine specimen in infancy, but its difficult to do!Pink urine due to uric acid crystals may be seen in diapers

IN-CLASS QUESTION***IN von GIERKES 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 firstCTP is synthesized from UMPPyrimidine ring synthesis completed first; then attached to ribose-5-phosphateN1, C4, C5, C6 : AspartateC2 : HCO3-N3 : Glutamine amide Nitrogen

Pyrimidine Synthesis

UMP Synthesis Overview2 ATPs needed: both used in first stepOne transfers phosphate, the other is hydrolyzed to ADP and Pi2 condensation rxns: form carbamoyl aspartate and dihydroorotate (intramolecular)Dihydroorotate dehydrogenase is an intra-mitochondrial enzyme; oxidizing power comes from quinone reductionAttachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-PPPi splits off PRPP irreversibleChanneling: enzymes 1, 2, and 3 on same chain; 5 and 6 on same chain

OMP DECARBOXYLASE : THE MOST CATALYTICALLY PROFICIENT ENZYMEFINAL REACTION OF PYRIMIDINE PATHWAYANOTHER MECHANISM FOR DECARBOXYLATIONA HIGH ENERGY CARBANION INTERMEDIATE NOT NEEDEDNO COFACTORS NEEDED !SOME OF THE BINDING ENERGY BETWEEN OMP AND THE ACTIVE SITE IS USED TO STABILIZE THE TRANSITION STATEPREFERENTIAL TRANSITION STATE BINDING

UMP UTP and CTPNucleoside 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 animalsBacteria regulation at ATCase rxnAnimals regulation at carbamoyl phosphate synthetase IIUDP and UTP inhibit enzyme; ATP and PRPP activate itUMP 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 AciduriaCaused by defect in protein chain with enzyme activities of last two steps of pyrimidine synthesisIncreased excretion of orotic acid in urine Symptoms: retarded growth; severe anemiaOnly 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 PyrimidinesCMP and UMP degraded to bases similarly to purines DephosphorylationDeaminationGlycosidic bond cleavageUracil reduced in liver, forming b-alanine Converted to malonyl-CoA fatty acid synthesis for energy metabolism

Deoxyribonucleotide FormationPurine/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: REVIEWDNA composed of deoxyribonucleotides

Ribose sugar in DNA lacks hydroxyl group at 2 Carbon

Uracil doesnt (normally) appear in DNAThymine (5-methyluracil) appears instead

Formation of DeoxyribonucleotidesReduction 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 R2A Heterotetramer: (R1)2 and (R2)2 in vitro

RIBONUCLEOTIDE REDUCTASER1 SUBUNITThree allosteric sitesSpecificity SiteHexamerization siteActivity SiteFive redox-active SH groups from cysteines

R2 SUBUNITTyr 122 radicalBinuclear Fe(III) complex

Ribonucleotide Reductase R2 Subunit

Fe prosthetic group binuclear, with each Fe octahedrally coordinated Fes are bridged by O-2 and carboxyl gp of Glu 115Tyr 122 is close to the Fe(III) complex stabilization of a tyrosyl free-radicalDuring the overall process, a pair of SH groups provides the reducing equivalentsA protein disulfide group is formedGets reduced by two other sulfhydryl gps of Cys residues in R1

Chime ExerciseE. coli Ribonucleotide Reductase:

3R1R and 4R1R: R1 subunit1RIB and 1AV8: R2 subunitExplore 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 R2Tyr 122 residueThe binuclear Fe (III) complexThe ligands of the Fe (III) complex

Mechanism of Ribonucleotide Reductase ReactionFree RadicalInvolvement of multiple SH groupsRR is left with a disulfide group that must be reduced to return to the original enzyme

RIBONUCLEOTIDE REDUCTASEACTIVITY IS RESPONSIVE TO LEVEL OF CELLULAR NUCLEOTIDES:ATP ACTIVATES REDUCTION OFCDPUDPdTTP INDUCES GDP REDUCTIONINHIBITS REDUCTION OF CDP. UDPdATP INHIBITS REDUCTION OF ALL NUCLEOTIDESdGTP STIMULATES ADP REDUCTIONINHIBITS CDP,UDP,GDP REDUCTION

RIBONUCLEOTIDE REDUCTASECATALYTIC ACTIVITY VARIES WITH STATE OF OLIGOMERIZATION:WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE OF R1 (CATALYTICALLY INACTIVE MONOMER) CATALYTICALLY ACTIVE (R1)2WHEN 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 enzymeThioredoxin gets oxidized to disulfideOxidized Thioredoxin gets reduced by NADPH ( final electron acceptor)mediated by thioredoxin reductase

Thymine FormationFormed by methylating deoxyuridine monophosphate (dUMP) UTP is needed for RNA production, but dUTP not needed for DNAIf 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: dUTPase1DUD: 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 siteFind 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 THFOxidized to dihydrofolateOnly known rxn where net oxidation state of THF changesTHF Regeneration:DHF + NADPH + H+ THF + NADP+ (enzyme: dihydrofolate reductase)THF + Serine N5,N10-methylene-THF + Glycine (enzyme: serine hydroxymethyl transferase)

dUMPdTMPNADPH + H+NADP+SERINEGLYCINEREGENERATION OF N5,N10 METHYLENETETRAHYDROFOLATEDHFN5,N10 METHYLENE-THFTHFdihydrofolate reductaseserine hydroxymethyl transferasethymidylate synthase

dUMPdTMPNADPH + H+NADP+SERINEGLYCINEINHIBITORS OF N5,N10 METHYLENETETRAHYDROFOLATE REGENERATIONDHFN5,N10 METHYLENE-THFTHFdihydrofolate reductaseserine hydroxymethyl transferasethymidylate synthase METHOTREXATE AMINOPTERIN TRIMETHOPRIMFdUMPXX

Anti-Folate DrugsCancer cells consume dTMP quickly for DNA replicationInterfere 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 DEFICIENCYIN PURINE DEGRADATION, ADENOSINE INOSINEENZYME IS ADAADA DEFICIENCY RESULTS IN SCIDSEVERE COMBINED IMMUNODEFICIENCYSELECTIVELY KILLS LYMPHOCYTESBOTH B- AND T-CELLSMEDIATE MUCH OF IMMUNE RESPONSEALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB ACTIVE SITE

Adenosine DeaminaseCHIME Exercise: 2ADAEnzyme catalyzing deamination of Adenosine to Inosine a/b barrel domain structureTIM Barrel central barrel structure with 8 twisted parallel b-strands connected by 8 a-helical loopsActive site is at bottom of funnel-shaped pocket formed by loopsFound in all glycolytic enzymesFound 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 DEFICIENCYONE OF FIRST DISEASES TO BE TREATED WITH GENE THERAPY

ADA GENE INSERTED INTO LYMPHOCYTES; THEN LYMPHOCYTES RETURNED TO PATIENT

PEG-ADA TREATMENTSACTIVITY LASTS 1-2 WEEKS