Test 4 GUTS Purines and Pyrimidines

What happens to cytosine? What is it degraded to?

Cytosine is deaminated, forming uracil, which is converted to CO2, NH4 and β-alanine.

OMP is decarboxylated by _____ to form ___. Why is this important?

OMP decarboxylase; UMP (uridylate); The first standard pyrimidine nucleotide made that is used for macromolecular biosynthesis.

What is the relationship between OPRTase and OMP decarboxylase? Why is this important? What are some outcomes?

OPRTase and OMP decarboxylase occur on a single protein known as UMP synthase. A hereditary defect in either activity leads to orotic aciduria.

Salvage of pyrimidines: U and O bases are salvaged to UMP and OMP by ___ part of the UMP synthase enzyme of pyrimidine synthesis.

Orotate phosphoribosyl transferase (OPRT)

Hyperuricemia can result from the control enzyme of purine synthesis...

Overproduction of purines from de novo synthesis - Overproduction of PRPP (mutation etc) stimulates Gln PRPP amidotransferase activity - Decreased utilization of PRPP (partial HGPRT deficiency reduces salvage consumption of PRPP etc) stimulate Gln PRPP amidotransferase activity

PNP deficiency. Inheritance? Characterized by?

PNP deficiency (autosomal recessive) results in immunodeficiency that is less severe than ADA-SCIDs, affecting only T-cells. Characterized by recurrent infections and neuro-developmental delays.

Purine synthesis begins with the synthesis of ___ by ____

PRPP (5 phosphoribosyl 1 pyrophosphate); PRPP synthetase

Salvage of purine bases uses ___ to form nucleotides. ___ for adenine to AMP.

PRPP; APRT

The R5P component of both purine and pyrimidine nucleotides comes through ___, made from ___ and ___ by ____.

PRPP; ATP and R5P

The committed and control step of purine synthesis is activated by ____ and inhibited by ___. What is von Gierke's disease?

PRPP; purine nucleotides (AMP etc.); Von Gierke's results in high levels of R5P which simulates PRPP formation and overrides this inhibition resulting in more purine nucleotides which are catabolized to uric acid. A contributing factor in the hyperuricemia and gout in these individuals.

How does trimethoprim work?

Trimethoprim, a folate analog, selectively inhibits bacterial DHR and is used as an anti-bacterial.

Gln donates ______; Asp donates ____; Gly donates ____

amino groups twice; one amino group; its entire backbone

Hyperuricemia can result from foods that are high in nucleic acid like ___... Most common cause of hyperuricemia?

animal liver can significantly increase blood and tissue urate levels Inefficient excretion of uric acid

What are the potential fates of (d)AMP? (d)IMP/(d)GMP/(d)XMP?

dAMP can be deaminated by AMP deaminase to dIMP or depend by 5nucleotidase to adenosine. (d)IMP/(d)GMP/(d)XMP are depend by 5nucleotidase to deoxy forms. (dI,dG,dX)

In PNP deficiency, dATP inhibition of ribonucleotide reductase leads to....

dATP inhibition of ribonucleotide reductase in the thymocytes leads to T-cell depletion. It has also been reported that the accumulation of deoxyguanosine triggers apoptosis in T cells, providing another mechanism for T-cell depletion. This does not affect the B cells in this disorder.

What happens to dTMP? Why is TTP important?

dTMP is sequentially phosphorylated to TTP needed for DNA synthesis. Note: TTP regulates ribonucleotide reductase specificity, switching its substrate specificity to purine NDP reduction (see above).

In SCID deoxyadenosine accumulates causing...

deoxyadenosine accumulates and adeosine kinase can convert it to dAMP then to dATP > accumulation of dATP > down regulation of rib nucleotide reductase > cells can no longer make dNTPs for DNA synthesis > stem cells are stimulated by cytokines to grow and differentiate into mature immune cells but they can't and die

PRPP synthetase is feedback _____ by ____ and ___ individually and synergistically

inhibited; GDP; ADP

What phosphorylates dNDPs to dNTPS?

nucleoside disphosphate kinase

nucleotide

nucleosides plus phosphate groups in ester linkage to 5' carbon of pentose sugar (NXPs)

Dihydroorotate dehydrogenase forms ___ in ___. Why is this important?

orotate; mitochondria; This is the only step not in the cytosol.

when base attached to ribose... attached to deoxyribose

ribonucleoside deoxyribonucleoside

what does purine nucleoside phosphorylase (PNP) do?

splits inosine, xanthosine and guanosine to R1P and hypoxanthine, xanthine, and guanine respectively

Functions of nucleotides:

substrates for DNA synthesis and repair RNA synthesis (transcription) carriers of high energy groups (phosphoryl, sugar moieties, basic moieties, acyl groups, methyl groups) Components of coenzymes (NAD, NADP, FAD, CoASH) signaling molecules allosteric regulators of enzymatic activity (cAMP)

Guanine is deaminated to ___ by ____. Why is this important?

xanthine; guanase (guanine deaminase). The pathways for the degradation of adenine and guanine merge at this point.

__ is the regulated step in mammals. What activates and inhibits?

CPSII; PRPP; UTP

CPSII step is analogous to first reaction in urea cycle catalyzed by __ in the mitochondria except CPSII is ___ and uses ___ instead of NH4 as a nitrogen donor.

CSPI; cytosolic; Gln

CTP feedback inhibits ____. What does this help balance?

CTP synthetase; UTP and CTP levels

___ is derived from UMP. UMP is sequentially phosphorylated to ___ and ___.

CTP; UDP; UTP

Pyrimidine synthesis begins when ___ combines with Glu with ___ and ___ to form _____.

Carbamoyl phosphate synthetase II; bicarbonate; ATP; carbamoyl phosphate.

ADA deficiency

Causes SCID.

Salvage enzymes do what?

Convert nucleobases into nucleosides and then to NTPs. Some tissues such and organs such as brain and bone marrow mainly synthesize nucleotides by a salvage pathway.

HGPRT: hypoxanthine or guanine converted to __ or __.

IMP or GMP

How are purines and pyrimidines synthesized differently with regards to the rings/ribose sugar?

In purine synthesis the ring is built upon the ribose but in pyrimidine synthesis the ring is completed and then connected to ribose.

Methotrexate does what? What is it used as a treatment for?

Inhibits dihydrofolate reductase, the enzyme that regenerates THF. Lowering THF limits purine synthesis as well as TTP synthesis. MTX is used in chemo to slow cancer cell growth but is toxic to all dividing cells.

What is the result of a defective HGPRT?

Lesch-Nyhan Syndrome (Xlinked) is the inability to salvage Hx or G. It results in an increased PRPP and decreased IMP/GMP. Glutamine PRPP amidotransferase (control of purine) has excess PRPP which overrides feedback inhibition --> purine synthesis is hyper active. Ineffective salvage combined with increased purine synthesis results in purines for degradation to uric acid. This causes hyperuricemia leading to urolithiasis and gouty arthritis. Present with severe neurological features including spasticity, mental retardation and self-mutiliation.

Since TTP is required for DNA replication this pathway is targeted to slow cell growth of cancer cells. What are methotrexate and aminopterin?

Methotrexate and aminopterin (folate analogs) inhibit DHR, reducing the amount of FH4 and the conversion of dUMP to TMP as well as de novo purine synthesis (N10-formyl-THF is utilized as a one carbon during purine synthesis). Toxic to all dividing cells -> side effects of chemotherapy (alopecia, myleosuppression, mucositis).

Dihydroorotase converts what into what?

N-carbamoyl aspartate into dihydroorotate.

How does megaloblastic anemia/B12 deficiency affect DNA synthesis?

N5-mTHF becomes THF in the methionine synthase (MS) conversion of homocysteine to methionine. Dietary vitamin B12 (cobalamin) is required for this reaction to proceed. The Vit. B12-dependence of this conversion of N5-mTHF to THF explains the megaloblastic anemia associated with a Vit. B12 deficiency. The deficiency prevents return of N5-mTHF to the THF pool. This decreases the THF available for N5, N10-mTHF formation. Eventually all of the folate may become trapped in the N5-mTHF form. The only available folates are derived from dietary intake. Decreased amounts of the THF forms will reduce de novo purine and dTMP biosynthesis, resulting in the anemia as blood stem cell replication is restricted.

What kind of enzymes interconvert NDPs and NTPs? Does it matter if it's ribose or deoxyribose?

Nucleoside diphosphate kinase. It is a single enzyme with a broad specificity for NDPs. (will do both GDP>GTP and ADP and CDP to ATP and CTP. Doesn't matter the sugar

Adenosine deaminase (ADA) converts __ to ___.

(d)adenosine to (d)inosine

Treatment for hyperuricemia (Gout): ELITEK. How is it made and what does it do? Do humans make urate? Is it long term or temporary?

- a recombinant Urate oxidase degrades urate to allantoin, which is 5-10X more soluble than urate. Produced as a recombinant protein in yeast cells from a cDNA obtained from a species of aspergillus (a mold). - Humans do not make urate oxidase - Intravenous protein drug for temporary relief of high plasma urate levels, as produced during some cancer chemotherapy.

Treatment for hyperuricemia (Gout): Allopurinol Is oxidized by what?

- oxidized by xanthine oxidase to alloxanthine (oxypurinol), an irreversible, suicide inhibitor of xanthine oxidase. - Uric acid levels fall and hypoxanthine and xanthine levels rise. - amount of purine being degraded is spread over three products rather than only one >> none of the compounds exceeds its solubility constant, precipitation does not occur, symptoms of gout gradually subside. - decreases the overall synthesis of purine nucleotides as Hx and X are salvaged by HGPRT using PRPP which reduces cellular PRPP and Gln PRPP amidotransferase activity

GTP activates/inhibits what?

---ACTIVATES: 1) AMP synthesis (cross-activate) 2) Activates Pyrimidine synthesis (want purines/pyrimidines to be balanced) ---INACTIVATES: 1) PRPP synthetase

Reducing reactions produce an oxidized RR enzyme which must be reduced to continue producing dNDP. How does this occur?

1. 2 e- (Hs) are transferred to RR catalytic site's disulfide bond from e- donor protein thioredoxin. This oxidizes thioredoxin. 2. Using NADPH, thioredoxin reductase converts oxThioredoxin to reduced form. 3. This series of reduction reactions requires sufficient cellular levels of NADPH for cells to commit to dNTP formation and DNA replication.

What are the outcomes of orotic aciduria? What is given orally to treat?

1. Autosomal recessive disorder > pyrimidines cannot be synthesized and growth retardation occurs. 2. Megaloblastic anemia (normal B12 and folate, does not respond to either) Oral administration of uridine bypasses metabolic block (salvage pathways allow uridine to be converted to UMP) providing source of pyrimidines and feedback inhabit of CPSII to slow down production of orotic acid.

UMP synthase is composed of what?

1. OPRTase 2. OMP decarboxylase defect in either = orotic aciduria

Regulation of RR. How many allosteric sites? What do they do?

1. Primary: controls overall activity (on/off). ATP stimulates, dATP inhibits. 2. Substrate specificity site: controls which NDP utilized as the substrate to produce a balanced level of dNTPs for DNA synthesis.

Since TTP is required for DNA replication this pathway is targeted to slow cell growth of cancer cells. What is 5-fluorouracil? 5FU

5-fluorouracil (5-FU) (and other thymine analogs) is metabolically converted by salvage enzymes to 5-FdUMP. 5-FdUMP is a suicide inhibitor of thymidylate synthase.

Uric acid has a pK of ___ and is not very soluble in an aqueous environment. It is ionized to ___ which is more soluble than uric acid. What happens to uric acid and __? iThe rate of uric acid excretion by the normal adult human is about ___ g/24 h, arising in part from ingested purines and in part from the turnover of the purine nucleotides of nucleic acids. iThe normal concentration of uric acid in the serum of adults is in the range of ___ mg/dl.

5.4; urate; both are excreted in urine .6 3-7

AMP is derived from IMP in what two steps?

6-O of inosine is displaced by Asp to yield adenylosuccinate (uses GTP) adenylosuccinate then carries out the nonhydrolytic removal of fumarate from adenylosuccinate leaving AMP

Purine synthesis: purine rings are assembled from precursors like __, ___ and ____

AAs (Gln, Asp, Gly); THF; CO2

IMP is a branch point for the production of __ and __.

AMP; GMP

Interconversion of nucleoside mono, di and trip phosphates: What is usually the energy donor? Are these reversible or irreversible? What kind of enzymes interconvert (d)NMPs and (d)NDPs? Do they discriminate between ribose and deoxyribose?

ATP; reversible; Base-specific nucleoside monophosphate kinases; No they do not discriminate between ribose and deoxyribose. (UMP kinase UMP > UDP, GMP kinase GMP > GDP) EXCEPTION: TMP kinase only accepts deoxyribose!

Hyperuricemia can result from increased cell death... What is Tumor lysis syndrome?

As a result of radiation therapy or cancer chemotherapy may elevate uric acid levels and lead to hyperuricemia. Tumor lysis syndrome occurs when large numbers of neoplastic cells are killed rapidly, leading to the release of intracellular ions and metabolic byproducts into the systemic circulation. Characterized by rapid development of hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia, and acute renal failure.

Pyrimidine rings are assembled from ___ ___ and ___.

Asp, Glu, CO2

ATCase adds an entire ___ molecule to carbamoyl phosphate to form ___. This enzyme is the control step in bacteria inhibited by ___. Is it regulated in humans?

Aspartate; N-carbamoyl-Aspartate; CTP; Not regulated in humans

Pyrimidine degradation leads to water soluble compound (ex?) that are excreted in urine. What is notable about pyrimidine degradation?

B-amino acids, CO2, NH4 (urea) There are no significant metabolic problems associated with degradation of pyrimidines.

SCID treatments?

Bone marrow transplant if matching donor available. ADA-SCID has been treated by gene replacement therapy.

How is the supply of adenine and guanine ribonucleotides balanced? Two ways.

Both GMP and AMP inhibit the first step in their own synthesis from IMP GTP is a substrate in AMP synthesis, and ATP Is a substrate in GMP synthesis.

What happens to excess nucleotides that are not salvaged?

Excess nucleotides that are not salvaged are dePed and the nucleosides are cleaved to produce R1P and free pyrimidine bases (cytosine, uracil, and thymine).

Purine synthesis requires __. How does this control purine synthesis?

FH4; reducing the level of FH4 will slow purine synthesis.

Nucleosides and dNucleosides are salvaged by ___. They use ___ as the phosphate donor. Thymidine kinase uses only __ as substrate. Adenosine kinase has a high ___ like AMP kinase, to maintain ATP levels.

Kinases; ATP; dT; efficiency

How is purine degradation done?

Leads to the production of uric acid which is secreted in urine. Sequential actions of nucleases and nucleotidases lead to the hydrolysis of nucleic acids to nucleosides.

What does ribonucleotide reductase do? (RR) What are the substrates?

Formation of deoxyribonucleotides required for DNA synthesis involves the reduction of the sugar moiety of NDPs by RR during the S-phase of the cell cycle. ONLY NDP forms are substrates and the products are dNDPs.

___ allosterically stimulates CTP syntheses. What does this balance?

GTP; Pyrimidine (GTP) and purine (CTP) levels Think DNA base pairings

How is RR activated? How is R2 degraded.

Gene encoding R2 subunit of RR is turned on late in G1 into S phase. RR is then active. With completion of DNA replication, transcription of R2 gene is turned off and existing R2 protein is degraded.

CTP synthetase adds and amino group from ___ to C4 of ___ to produce CTP using ATP as energy. This conversion only occurs for the ___ forms.

Gln; UTP; triphosphate

What does hydroxyurea do?

Hydroxyurea scavenges the free radical on the tyrosine in the catalytic site of RR and is used to reduce the rate of cell division in cancer therapies. Hydroxyurea is also used in some sickle cell therapies to induce hemoglobin γ-chain synthesis so fetal hemoglobin can replace hemoglobin S.

What is the first purine base made? What are the names of its nucleoside and nucleotide forms?

Hypoxanthine (Hx); inosine; IMP

Xanthine oxidase converts ___ >>> ____ >>> _____.

Hypoxanthine >> xanthine >> uric acid

GMP is derived from IMP in what two steps?

IMP dehydrogenase catalyzes the oxidation of IMP at C2 yielding XMP the amido N of glutamate is donated to the C2 position of XMP yielding GMP (ATP used)

Only a small amy of dietary bases and nucleosides are absorbed into the circulation and then salvaged in tissues, most are degraded. Purines are degraded into what where? Pyrimidines are degraded where?

Purines into urate in intestinal mucosa. Pyrimidines in liver.

Orotate phosphoribosyl-transferase (OPRT) attaches the ___ from PRPP to rotate to form what?

R5P; Forms the first pyrimidine ribonucleotide orotidylate (OMP).

Why salvage nucleosides and free bases released by nucleic acid degradation, DNA or RNA turnover?

Salvage consumes less energy than de novo synthesis and is energetically preferred. The ability to salvage nucleotides from sources in the body essentially alleviates nutritional requirement for nucleotides.

Hyperuricemia?

Serum urate level >7 mg/dl either prolonged or acute elevation of blood urate, can lead to precipitation of monosodium urate crystals in the synovial fluid of joints (big toe is commonly affected) and in soft tissues. This leads to inflammation and painful arthritis (Gout) and, over time, to joint degradation. Crystals can also form in the kidney (urolithiasis), leading to renal damage.

How is thymine different?

Thymine is salvaged by thymidine phosphorylase to the deoxynucleoside form (dT), NEVER to a nucleoside. TTP is then generated by thymidine kinase and nucleoside diphosphate kinase.

What must happen to sustain dTMP formation for ongoing DNA replication?

To sustain dTMP formation, DHF must be reduced to THF by dihydrofolate reductase (DHR). THF is then methylated by serine hydroxymethyltransferase to reform N5,N10-mTHF.

In SCID T and B cells...

T and B cells are deficient >> no functional immune system >> death from infection

The next nine reactions in purine synthesis form the purine ring using ___, ___, and ___ as building materials. We don't memorize these!

THF, Asp, Gly

What is the committed and control step of purine synthesis? What is it catalyzed by?

The formation of 5-phosphoribosyl-1-amine catalyzed by glutamate PRPP amido transferase

What do sulfonamides do?

They inhibit bacterial folate synthesis. Since humans do not synthesize folate (it's in the diet), this does not affect purine synthesis/folate in humans.

____ catalyzes the formation of dTMP by methylating dUMP. A 1C unit from ____ is transferred to C5 of uracil ring and thus it is oxidized to ___.

Thymidylate synthase; N5/N10 mTHF; DHF

What happens to thymine? What is it degraded to? What is important about this compound?

Thymine is converted to CO2, NH4 and β-aminoisobutyrate. Since β-aminoisobutyrate originates exclusively from thymine degradation its presence in urine is an indicator of DNA turnover. (Can be elevated during chemotherapy or radiotherapy).

In PHP deficiency, guanosine accumulates....

With a PNP deficiency, guanosine will accumulate which inhibits the conversion of dGMP to guanosine via the actions of 5′-nucleotidase. As dGMP accumulates, it will be phosphorylated to form dGTP. Concurrently, inosine will accumulate, slowing the conversion of dAMP to inosine and leading to an increase in dATP levels.