BCM 32: Synthesis of Purines & Pyrimidines

Orotic aciduria can also occur as a result of the deficiency of _____, a Urea cycle enzyme Lack of OTC causes an accumulation of carbamoyl phosphate in mitochondria which then spills over into the cytosol In cytosol, large amounts of carbamoyl phosphate enter the pyrimidine synthesis pathway and cause a build up of intermediates, particularly orotic acid

Ornithine transcarbamoylase (OTC)

What disorder: Causes retarded growth, leukopenia, and severe anemia (hypochromic erythrocytes and megaloblastic bone marrow) What is the Tx?

Orotic aciduria →Loss of both RBC and WBC uridine and/or cytidine

What E regulates Purine Synth? Note: GMP and AMP FB inhib its own synth (for balance of GTP and ATP synth)

PRPP Amidotransferase

_____ Connects Carbohydrate and Nucleotide Metabolism: ______ is essential for de novo as well as salvage pathways of synthesis of both purines and pyrimidines

PRPP Connects Carbohydrate and Nucleotide Metabolism: PRPP

Know: Ribose-5-P + ATP→ 5-phosphoribosyl-1-pyrophosphate(PRPP) + AMP What enzyme?

PRPP Synthetase

Must know: State what level each is formed at: Cytidine nucleotides are formed at the level of __________ Deoxyribonucleotides are formed at the level of ________ Deoxythymidine nucleotides are formed at the level of _________

Tri di mono

What inhibits ribonucleotide reductase?

dATP (& hydroxyurea)

Compounds That Interfere With Nucleotide Synthesis Are Important Drugs Antifolates interfere with folate → dihydrofolate → tetrahydrofolate →Can't make ____ →Can't make _____ Base/nucleoside analogs interfere with nucleotide synthesis (antimetabolites) →Anticancer drugs →Antiviral drugs

dTMP purines

Thymidylate synthesis

dUMP→dTMP

Synthesis of Deoxyribonucleotides: Deoxyribonucleotides are made at the level of... _________ is a small redox protein that is a cofactor for ribonucleotide reductase (used NADPH as a cofactor) _____ is the major regulator of dNDP production

nucleoside diphosphates (NDPs) Thioredoxin dATP...dNDP

Pyrimidine synthesis occurs where?

occurs in a variety of tissues

Salvage Pathway of Nucleotide Synthesis Mainly involves ____________ of a free purine by ______ Involves 2 enzymes: 1) 2) Deficiency of HGPRT results in the genetic disorder= Pyrimidine phosphoribosyl transferase similarly derives CMP, UMP, TMP

phosphoribosylation PRPP 1) Adenine phosphoribosyl transferase (APRT) 2) Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Lesch-Nyhan syndrome

X-linked recessive >200 mutations found Diagnosis: <1.5% HGPRT activity = inability to salvage hypoxanthine or guanine State if inc or dec: __PRPP; __IMP; ___GMP hyper_______with gout →Uric acid is a product of purine degradation

↑PRPP; ↓IMP; ↓GMP Uricemia

Anticancer Drugs: Treats acute lymphocytic leukemia, needs activation to nucleoside monophosphate, inhibits conversion of ____ to ____ and GMP (de novo) Treats colon (and other) cancers, needs activation (salvage pathway) to nucleotides F-UTP and F-dUMP which inhibit RNA processing and thymidylate synthase, respectively. Treats leukemias/non-Hodgkin lymphoma, phosphorylated to nucleotide, inhibits DNA polymerases.

IMP to AMP

State if a + or - regualtor: AMP, GMP IMP PRPP 6-Merctopurine nucleotide Allopuritinol Nu

Inhibitor Activator Inhibitor Inhibitor

Motor dysfunction; hypotonia; self-mutilation; mental retardation =What disease?

Lesch-Nyhan Syndrome

Purine synthesis Purine de novo synthesis is most active where? Non-hepatic tissues →Generally have limited or absent de novo purine synthesis →Rely heavily on pre-formed bases (Salvage Pathway) from own intracellular turnover →Bases synthesized in the liver and delivered via the blood

Liver

__________ needed for purine synthesis ___________ needed for conversion of dUMP to dTMP

N10-Formyl-THF N5,N10-Methylene-THF

Overview of Purine Synthesis: 1. Ribose-5-P 2. PRPP 3. 5-phosphoribosylamine 4. IMP 5. GMP or AMP GMP has an amino from what AA? AMP has an amino from wha AA?

*Ignore intermediate steps *Don't find IMP in RNA or DNA (maybe tRNA) Glutamine Aspartate

Name the activated carriers: 1. Sugars 2. Head groups (choline, ethanolamine) 3. Methyl donors 4. Sulfur donors

1. UDP 2. CDP 3. SAM 4. PAPS (Phopho-adenosyl-phosphate-sulfate)

Regulation of De Novo Pyrimidine Synthesis CPS II →UTP →CTP →ATP →PRPP ATCase →CTP →ATP UMP synthase →UMP CTP synthetase →CTP

CPS II Inhibited by UTP and CTP Activated by ATP and PRPP ATCase Inhibited by CTP Activated by ATP UMP synthase Inhibited by UMP CTP synthetase Inhibited by CTP

Regulation of Purine Synthesis In addition, feedback inhibition of PRPP synthetase by ___ and ____

ADP and GDP IMP GMP AMP All neg. regulate that step

Enzymes of Pyrimidine Synthesis CAD - cytosolic polypeptide that contains the first 3 enzyme activities involved in de novo pyrimidine nucleotide synthesis: C? A? D? The only enzyme of pyrimidine biosynthesis that is not cytosolic is? →UMP synthase is a single polypeptide with two enzymatic activities 1) 2)

C − Carbamoyl phosphate synthetase II A − Aspartate transcarbamoylase D − Dihydroorotase Dihydroorotate dehydrogenase →→Orotate phosphoribosyltransferase →→OMP decarboxylase

What are the 3 sources of atoms in a pyrimidine ring? What's the primary source?

CO2 (1 C) Gln (1 N) Asp (MAIN) (3 C's + 1N)***

Acyclovir 2-azido-3'-deoxythymidine

Antiviral drugs

Which of the following molecules is the major contributor of atoms that make up the ring of pyrimidines?

Aspartate

______ is a Suicide Inhibitor of Thymidylate Synthase

F-dUMP Flourodeoxyirudylate+ N5,N10-methylene tetrahydrofolate → Stable adduct (deoxyribose-P)

Objective 2: Describe the salvage pathway of synthesis of nucleotides and the Lesch-Nyhan syndrome

FYI: A salvage pathway is a pathway in which nucleotides (purine and pyrimidine) are synthesized from intermediates in the degradative pathway for nucleotides. Salvage pathways are used to recover bases and nucleosides that are formed during degradation of RNA and DNA.

What enzyme derives CMP, UMP, TMP ?

Pyrimidine phosphoribosyl transferase

_________=Inhibit Folate synthase (Prokaryotes) _________= Inhibits Dihydrofolate Reductase (Prokaryotes) __________= Inhibits Dihydrofolate Reductase (Protozoans) _________=Inhibits Dihydrofolate Reductase (Eukaryotes) →Anticancer drug (acute leukemia)

Sulfonamides Trimethoprim Pyrimethamine Methotrexate

Rate-limiting and Committed Step of Purine Biosynthesis

The synthesis of Phosphoribosylamine by glutamine:PRPP amidotransferase is the rate-limiting and committed step The most regulated step

Genetic deficiency of __________ (a bifunctional enzyme with 2 activities - Orotate phosphoribosyltransferase (O-PRT) and OMP decarboxylase Less UMP → less ____ → less feedback inhibition of _____ → more _____ production

UMP synthase UTP CPS-II Orotate

Hyperammonemia is seen in OTC deficiency but not in .... Leukopenia and megaloblastic anemia seen in OMP synthase deficiency but not in ....

UMP synthase deficiency OTC deficiency *** see summary table in slides

UTP vs. CTP Which is made at the triphosphate level? Which is made at the monophosphate level?

UTP=Mono CTP=Tri

UMP→UDP via what E? UDP→UTP via what E?

Uridylate Kinase Nucleoside diphosphate Kinase

Where does de novo pyridine synth occur? What is made first? Followed by what?

Variety of tissues Ring made first Then, attached to R5P (Ribose) (Opposite in Purine synth)

Sources of Purine Ring Atoms Purine ring is built on what foundation? Name the 5 components of the purine ring atom 1. 2. 3. 4. 5. Which provides the most C's?

activated ribose 5-phosphate 1. Asparatate (N) 2. CO2 (C) 3. Glycine (C-C-N) 4. Glutamine (N) 5. N10-Formyl tetrahydrofolate (2 C's) Glycine! (C-C-N)