Nucleotides: Synthesis and Degradation

Nitrogenous Bases Planar, aromatic, and heterocyclic Derived from purine or pyrimidine Numbering of bases is “unprimed”

Nucleic Acid Bases Pyrimidines Purines

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: Nucleotides: 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 Metabolism PURINE 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 Amine C2, C8: Formate N3, N9: Glutamine C4, C5, N7: Glycine C6: Bicarbonate Ion

Purine Nucleotide Synthesis

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 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 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 CYCLE 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

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 + PRPP  AMP + PPi 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”

ALCOHOL CONSUMPTION AND GOUT 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

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

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 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 b-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

E. coli Ribonucleotide Reductase: Chime Exercise E. coli Ribonucleotide Reductase: 3R1R and 4R1R: R1 subunit 1RIB 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

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)

REGENERATION OF N5,N10 METHYLENETETRAHYDROFOLATE dUMP dTMP thymidylate synthase DHF N5,N10 – METHYLENE-THF NADPH + H+ GLYCINE dihydrofolate reductase serine hydroxymethyl transferase NADP+ SERINE THF

INHIBITORS OF N5,N10 METHYLENETETRAHYDROFOLATE REGENERATION dUMP dTMP thymidylate synthase DHF N5,N10 – METHYLENE-THF X NADPH + H+ GLYCINE FdUMP dihydrofolate reductase serine hydroxymethyl transferase NADP+ SERINE X THF METHOTREXATE AMINOPTERIN TRIMETHOPRIM

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