M2O Purines and Pyrimidines

Outline, in broad strokes, the synthesis of dADP from IMP including regulation

1) IMP --> AMP Add nitrogen from aspartate 2) AMP --> ADP add phosphate using AMP (adenylate) kinase Requires GTP (ensures equal amounts of ATP and GTP Inhibited by AMP (product) 3) ADP--> dADP using ribonucleotide reductase enzyme is activated by ATP substrate and dGTP (ensuring equal amounts of dGTP and dATP) inactivated by dATP product

List the origin of the ring atoms in pyrimidine and the order of their addition

1) N from glutamine 2) C from respiratory CO2 3) aspartate

Describe the pathologies discussed in salvage pathways (ADA deficiency)

Adenosine deaminase (ADA) deficiency -ADA is enzyme used in adenosine breakdown -dATP usually inhibits ribonucleotide reductase RNR during purine synthesis -without ADA there is no adenosine breakdown and excess dATP inhibiting RNR -Leads to developmental arrest and apoptosis of lymphocytes -Results in severe combined immunodeficiency syndrome ADA-SCID

Name the nucleoside and nucleotide from each base

Base: Adenine Cytosine Guanine Thymine/Uracil Nucleoside: (Deoxy)Adenosine (Deoxy)Cytidine (Deoxy)Guanosine Deoxythymidine/Uridine Nucleotide: Nucleoside 5 Monophosphate (d)AMP (d)CMP (d)GMP dTMP/UMP

What is meant by CAD? Which domain is regulated? How is it regulated?

CAD is a trifunctional protein on which the first 3 reactions of pyrimidine synthesis occurs Carbamoyl phosphate synthetase II (CPS II) is regulated This facilitates the first step of getting N from glutamine Activated by PRPP and inhibited by UTP

What is the importance of dUTPase

Changes dUTP to dUDP and dUMP

Describe the pathologies discussed in salvage pathways (Lesch-Nyhan Syndrome)

HGPRT deficiency would result in increased PRPP concentrations which promotes de novo synthesis of purines When those purines are not needed they get degraded thus causes increased uric acid This leads to gout, neurological abnormalities such as self mutilation, aggressive behavior, developmental delay, motor impairment, and renal failure Neuro problems from HGPRT deficiency highlights importance of salvage in CNS

What is the importance of methotrexate

Methotrexate is a folic acid analog drug that inhibits DHF reductase

What is the relationship between urate concentration and a) PRPP synthetase activity

Mutations in PRPP synthetase that increase it's activity increase urate concentration

Describe the role PRPP plays in de novo synthesis and salvage

PRPP is the sugar phosphate starting block that the purine is build on in de novo synthesis PRPP provides the phospho ribosyl (sugar phosphate) group in salvage. PRPP used in salvage process inhibits de novo synthesis because it reduces its cytosolic concentration and thus theres less available for use in that pathway

What is the relationship between urate concentration and b) HGPRT activity

Partial HGPRT deficiency leads to primary gout and total HGPRT deficiency leads to secondary gout. Problems with this enzyme means theres no salvage pathway competing with de novo synthesis so excess purines get formed and uric acid builds

Distinguish primary gout from secondary gout

Primary: caused by excess PRPP due to mutations in PRPP synthetase that causes increased activity in enzyme. also caused by Partial HGPRT deficiency causing downregulated salvage pathway Secondary: gout is secondary result of a different disorder total HGPRT deficiency increased cell turnover in cancer Impaired renal excretion from lead poisoning Increased conversion of ATP to AMP due to alcohol consumption, hypoxia, glycogen storage disease, fructose metabolism errors

In what pathway does adenosine deaminase function? What are some consequences of a deficiency in this enzyme?

Purine catabolism ADA-SCID

Distinguish the structure of a purine from a pyrimidine, a base from a nucleoside, and a nucleoside from a nucleotide

Purine is two rings: Adenine, Guanine, Xanthine, Hypoxanthine Pyrimidines is one ring: Thymine, Cytosine, Uracil, Orotic Acid These ringed structures are bases Nucleoside is the base + ribose sugar Nucleotide is the base + sugar + phosphate groups

Describe the pathologies discussed in salvage pathways (PNP deficiency)

Purine nucleoside phosphorylase deficiency T cell defects (less severe than ADA)

List the origin of the ring atoms in purines and the order of their addition

Purine: 1) Amide from glutamine 2) Glycine residue 3) Carbon from N10-formyl-tetrahydro folate (THF) 4) Amide from glutamine 5) Carbon from Respiratory CO2 6) Nitrogen from Aspartate 7) Carbon from THF

Compare and contrast the de novo synthesis of purine and pyrimidine ribonucleotide triphosphates including physiologic and pharmacologic regulation. Describe the pathologies discussed

Purine: 1st base is xanthine Pyrimidine 1st base is orotic acid Purine ring is formed onto PRPP Pyrimidine ring is formed first then PRPP is incorporated

Compare and contrast the degradation of purine and pyrimidine ribonucleotide triphosphates including physiologic and pharmacologic regulation. Describe the pathologies discussed

Pyrimidine has no pathologies associated with degradation Purine has ADA deficiency, PNP deficiency, and Lesch-Nyhan syndrome

What is hereditary orotic aciduria? How is it treated? What does this suggest about our ability to salvage pyrimidines?

Pyrimidine nucleotide de novo synthesis disorder UMP synthase defect Orotate phosphoribosyl transferase facilitates orotic acid-->OMP Orotate decarboxylase domain facilitates OMP --> UMP Causes failure to thrive due to inability to replicate intestinal cells and anemia due to inability to replicate reticulocytes for RBC Treatment: Uridine Uridine --> UMP --> UTP UTP inhibits CPS II of de novo pyrimidine synthesis, so this shows that there is an intact salvage system

What is the importance of DHF reductase

Reduces DHF so it becomes THF again and can be used by thymidine synthase in TMP synthesis

What is the regulated step in pyrimidine synthesis in humans? How is it regulated? What is the committed step?

Regulated step: Synthesis of carbamoyl phosphate by carbamoyl phosphate synthetase II (CPSII) Activated by PRPP Inhibited by UTP Committed step: Synthesis of carbamoyl aspartate by aspartate transcarbamoylase

How is PRPP synthesized? How is its synthesis regulated?

Ribose 5 phosphate has more phosphate added to it from prpp synthase enzyme is inhibited by end products

What is meant by salvage pathways? What role does PRPP play? What is the significance of HGPRT in salvage?

Salvage pathways are shortcuts to regenerating nucleotides without going through the energy expensive 11 step process of purine synthesis again PRPP is used as a substrate in the salvage pathway (uses it's phospho ribosyl to add to base) HGPRT is enzyme

Recognize the functions of purines and pyrimidines

They serve as nitrogenous bases for DNA and RNA synthesis

What is the importance of 5-FU

Thymidylate synthase is inhibited by 5-fluorouracil (5-FU). This drug acts as an analog of thymine when added to PRPP. The deoxy form inhibits thymidylate synthase so TMP can't be formed

Distinguish the structure of thymine from uracil

Thymine is a methylated uracil

Explain the generation of the deoxyribose form from the ribose form, including physiologic and pharmacologic regulation

Uses Ribonucleotide reductase (RNR) activated by NTP substrate and dNTP (On specificity site positive allosteric effector based nucleotide being formed) Activated by ATP on activity site inactivated by dATP product on activity site Hydroxyurea drug inhibits ribonucleotide reductase (ADP--> dADP) used for cancer treatment to prevent rapid DNA synthesis of tumor

In de novo synthesis of pyrimidines, what is the source of each of the following? How is each generated? a) UMP b) CTP d) TMP

a) OMP is decarboxylated to form UMP with orotidylic acid decarboxylase b) UTP is aminated with glutamine to form CTP with CTP synthase c) dUMP/dUTP/dCMP is methylated with thymidylate synthase and THF to form TMP

What is the relationship between urate concentration and c) amidotransferase activity

amidotransferase is the committed step of de novo synthesis activating this will increase urate concentration inhibiting will decrease

Describe the risks associated with hyperuricemia

gout

What is the relationship between urate concentration and e) gout

high urate is cause of pain in gout

What is the relationship between urate concentration and d) use of colchicine, indomethacin, allopurinol, and probenecid

indomethacin: anti-inflammatory treatment Colchicine: steroid treatment allopurinol: Long term therapy xanthine oxidase inhibitor (catabolism enzyme) probenecid: long term therapy inhibits proximal tubule reuptake of urate

Outline the three requirements for gouty arthritis

surplus of uric acid crystal formation of urate in synovial joints Inflammatory response to crystals, causing bone destruction in some cases

What are the five general steps in the catabolism of purines? What is the final product in humans? What is the function of xanthine oxidase?

1. Nucleic acids --> Oligonucleotides with nuclease 2. Oligo --> nucleotides with phosphodiesterase 3. Nucleotides --> nucleosides with nucleotidase 4. Nucleosides --> ribose 1 phosphate and free base with nucleoside phosphorylase R1P-->R5P using mutase 5. Base -->hypoxanthine/guanine--> xanthine with purine nucleoside phosphorylase then xanthine oxidase or guanase Final product in humans: uric acid xanthine oxidase turns hypoxanthine to xanthine and then xanthine to uric acid for excretion

Outline the various treatment strategies for gout

1. antinflammatory (NSAIDS or analgesic , not steriods not aspirin) 2. Steroids: prednisone 3. Chronic therapy

Define the clinical stages of gout

1. asymptomatic hyperuricemia with intermittent attacks 2. Diagnosed with needle shaped crystals in fluid collected from joint or tophus 3. Without treatment it progresses into chronic tophaceous gout leading to destruction of joint, deformities, and severe loss of function Any stages may show uric acid stones

Which is the committed step of de novo purine synthesis? What enzyme is involved? How is the step regulated?

Adding nitrogen from glutamine to PRPP PRPP --> 5-phospho-beta-D-ribosylamine Enzyme: PRPP glutamyl amidotransferase Inhibited by end products Activated by PRPP (free PRPP can overcome inhibition from end products)

What is the importance of hydroxyurea

Drug that inhibits RNR which turns ribose into deoxys

What is the importance of thymidylate synthase

Enzyme turns dUMP/dUTP/dCMP into TMP Requires THF oxidation to DHF during reaction Inhibited by 5-fluorouracil (5-FU). This drug acts as a analog of thymine when added to PRPP. The deoxy form inhibits thymidylate synthase

What is a regulated step in purine synthesis that IS NOT the committed step? Which enzyme is used?

First step: ribose-5-phosphate --> PRPP Enzyme: PRPP synthetase Uses ATP -inhibited by end products (mostly AMP and GMP)