Identify each reaction catalyzed by a nucleotidase, a phosphorylase and a phosphoribosyltransferase.
Top left, phosphoribosyl transferase in both directions
top right, nucleotidase from GMP to guaninise
center bottom, phosphorylase in both directions
Each diagram below represents percent initial nucleotide level as a function of time after administration of one of the four agents (hydroxyurea, trimethoprim, thymidine or fluorodeoxyuridine), for each of the four nucleotides (semiquantitatively). Match
Effect of thymidine on dNTP levels: high dTTP
Effect of trimethoprin on bacterial rNTP levels: low GTP and ATP on same curve
Effect of fluordeoxyuridine on dNTP levels: high dCTP
Effect of hydroxyurea on dNTP levels: dCTP and dTTP on same curve, dATP and
Radioactive uracil can be used to label all of the pyrimidine residues in DNA.
Part A. Using either names or structures, complete pathways for the conversion of uracil to dTTP and to dCTP.
(1) Uridine + ATP > UMP + ADP
(2) dCMP + H2O > dUMP + NH3
(3) UTP + Glu + ATP > CTP + Glu + ADP + Pi
(4) Uracil + ribose-1-phosphate > Uridine + Pi
(5) CDP + 2[H] dCDP + H2O
(6) dUMP + 5,10-methylene-THF > dTMP + DHF
(7) dTMP + ATP > dTDP + ADP
(8) dTDP +
Radioactive uracil can be used to label all of the pyrimidine residues in DNA.
Part B. Match the cofactors to the reactions they are involved in and their effect on this reactions.
dCMP+H2O?dUMP+NH3dCMP+H2O?dUMP+NH3. Cofactor: dTTP Effect: Inhibition
CDP+[2H]?dCDP+H2OCDP+[2H]?dCDP+H2O. Cofactor: dGTP Effect: Inhibition
UTP+Gln+ATP?CTP+Glu+ADP+PiUTP+Gln+ATP?CTP+Glu+ADP+Pi. Cofactor: GTP Effect: Activation
Radioactive hypoxanthine (HX) can be used to label purine residues.
Part A. Using either names or structures, complete pathways for the conversion of hypoxanthine to dATP and dGTP.
AMP + ATP ==> 2ADP
XMP+Glu+ATP ==>GMP+Glu+AMP+Pi
ADP+[2H]==>dADP+H2O
HX+PRPP==>IMP+PPi
GMP+ATP==>ADP+GDP
IMP+Asp+GTP==>Adenylosuccinate + GDP + Pi
Adenylosuccinate==>AMP+Fumarate
IMP+NAD++H2O==>XMP+NADH+H+
dADP+ATP==>dATP+ADP
dGDP+ATP==>dGTP+ADP
GDP+[2H]=
Radioactive hypoxanthine (HX) can be used to label purine residues.
Part B. Match the cofactors to the reactions they are involved in and their effect on this reactions.
ADP+[2H]?dADP+H2OADP+[2H]?dADP+H2O. Cofactor: dGTP Effect: Activation
IMP+Asp+GTP?Adenylosuccinate+GDP+PiIMP+Asp+GTP?Adenylosuccinate+GDP+Pi. Cofactor: AMP Effect: Inhibition
GDP+[2H]?dGDP+H2OGDP+[2H]?dGDP+H2O. Cofactor: dTTP Effect: Activation
IMP+NAD++H
Inosine 5'-monophosphate, which contains the base hypoxanthine, is a common intermediate for ________ nucleotide biosynthesis.
purine
Formyltransferases, which are ________ dependent, are involved in several steps of purine nucleotide biosynthesis.
tetrahydrofolate
Leukemia is a neoplastic (cancerous) proliferation of white blood cells. Clinicians are currently testing deoxycoformycin, an adenosine deaminase inhibitor, as a possible antileukemic agent. Why might one expect this therapy to be effective?
Deoxycoformycin might lead to adenosine and deoxyadenosine accumulation, since their deamination would be inhibited, as in adenosine deaminase deficiency. This could cause dATP to accumulate thereby inhibiting all four activities of ribonucleotide reducta
Cell cultures can be synchronized, or brought into the same phase of the cell cycle, by various means. For example, adding thymidine to a cell culture causes all cells to become arrested early in S phase.
What is the mechanism by which thymidine treatment
Thymidine added to cell culture medium is taken up into cells and converted by salvage pathways to thymidine triphosphate. dTTP accumulation blocks DNA synthesis reversibly by allosteric inhibition of CDP reduction by ribonucleotide reductase. So thymidin
As stated in the text, bacteriophages have been discovered with the following base substitutions in their DNA.
Part A
Part complete
dUMP completely substitutes for dTMP. Choose a set of virus-coded enzyme activities that could lead to the observed substit
virus-specified inhibitor of thymidylate synthase
virus-specified inhbitor of dUTPase
As stated in the text, bacteriophages have been discovered with the following base substitutions in their DNA.
Part B
5-hydroxymethyl-dUMP completely substitutes for dTMP. Choose a set of virus-coded enzyme activities that could lead to the observed subst
virus-specified hydroxymethyl-dUMP kinase
virus-specified dUMP hydroxymethyltransferase
As stated in the text, bacteriophages have been discovered with the following base substitutions in their DNA.
Part C
Complete the balanced equation for this reaction.
dUMP > hmdUMP > hmdUDP > hmdUTP
1. 5,10-CH2-THF > THF
2. ATP > ADP
3. ATP > ADP
Part D
5-methyl-dCMP completely substitutes for dCMP
virus-specified dCMP methylase
Part E
Complete the balanced equation for this reaction.
dCMP > CH3-dCMP > CH3-dCDP > CH3-dCTP
1. 5,10-CH2-THF
2. ATP > ADP
3.ATP > ATP
Which of the following enzymes is NOT primarily used in salvage of nucleic acids because the pyrophosphate product is rapidly hydrolyzed?
Phosphoribosyltransferase
T or F? 5-phosphoribosyl-?-D-1-pyrophosphate is a key intermediate in de novo synthesis of nucleotides but not in salvage pathways.
False
Which of the following compounds is NOT a precursor for nucleotide biosynthesis?
Fumarate
Which intermediate or intermediates in purine nucleotide biosynthesis would you expect to accumulate in DON-treated cells?
PRPP
XMP
formylglycinamide ribonucleotide
Which participants in pyrimidine synthesis would you expect to accumulate in DON-treated cells?
UTP
The purinosome contains enzymes that convert the serine hydroxymethyl group to the formyl group of 10-formyltetrahydrofolate
1)Serine+THF?Glycine+5,10?methylene?THF+H2O2) 5,10?METHYLENE-THF+NAD(P).?5,10?methenyl?THF+NAD(P)H+H+3) 5,10?methenyl?THF+H2O?10?formyl?THF
Consider either the purinosome or a multifunctional protein such as the CAD protein. Choose the evidence showing that the enzyme "channels" substrates through a multistep reaction sequence.Select all that apply
1. The whole pathway would be immediately in
3.Intermediates in a pathway accumulate only to a low level, because they are rapidly transferred to the next enzyme in a sequence.
4.The product of a reaction sequence reaches its maximal rate of synthesis faster than in a nonchanneled pathway, because e
Choose the correct balanced equations for the three known reactions that transfer an amino group to a substrate by condensation with aspartate to give an intermediate that then undergoes an ??,??-elimination to give the product plus fumarate.
Part A
Citrulline+aspartate+ATP?argininosuccinate+AMP+PPi
IMP+aspartate+GTP?adenyloaspartate+GDP+PiIMP+aspartate+GTP?adenyloaspartate+GDP+Pi
4?carboxy?5?aminoimidazoleribonucleotide+aspartate+ATP?N?succinylo?5?aminoimidazole?4?carboxamideribonucleotide+ADP+Pi4?c
The text states that ATP is synthesized primarily by energy metabolism, whereas other nucleoside triphosphates are formed from the action of nucleoside diphosphate kinase. What additional pathway exists for GTP synthesis?
the succinyl-CoA synthetase reaction in the citric acid cycle synthesizes GTP from GDP
Which of the following statements about purine biosynthesis in vertebrates is FALSE?
1. The multienzyme complex is called a purinosome.
2. Six proteins form a multienzyme complex with covalent linkages.
3.Many of the proteins are multifunctional in that t
2. Six proteins form a multienzyme complex with covalent linkages
T or F?
The initial end products of purine nucleotide biosynthesis are monophosphates.
True
The text describes a form of gout that results from HGPRT deficiency. Which enzyme abnormalities might similarly lead to hyperuricemia?Select all that apply.
1.mutations that lead to overexpression of PRPP synthetase
2.mutations that weaken allosteric reg
1.mutations that lead to overexpression of PRPP synthetase
3. mutations that cause overexpression of xanthine dehydrogenase
4.mutations that weaken allosteric regulation of the conversion of PRPP to 5-phosphoribosylamine
Why might this treatment be effective, while injection of the missing enzyme is ineffective in other conditions, such as HGPRT deficiency or a deficiency of one of the mitochondrial deoxyribonucleoside kinases?
Injected adenosine deaminase need not be taken up into cells in order to be effective because the substrates, adenosine and deoxyadenosine, are uncharged and, when they accumulate, can efflux from cells and undergo deamination in the extracellular space a
T or F?
Nucleoside phosphorylase cleaves a glycosidic bond to yield a base and either ribose-1-phosphate or deoxyribose-1-phosphate.
True
The end product of purine catabolism in humans is:
allantoin
xanthine
uric acid
hypoxanthine
urea.
uric acid
CTP synthetase is allosterically activated by GTP. What function might this play in the cell?
1. This is a way to prevent GTP overproduction, because CTP, in its turn, is an allosteric inhibitor of purinosome enzymes E1 and E3 involve d in produxtion of G
3. This is a way to keep purine and pyrimidine nucleotide pools in balance.
In the pyrimidine degradative pathway, all pyrimidines undergo conversion to uracil, which undergoes an NADPH-dependent reduction.
Part A
Choose plausible reactions leading from cytidine, cytosine, and uridine to uracil.
Cytosine+H2O?uracil+NH3
Cytidine+H2O?uridine+NH3
Uridine+Pi?uracil+ribose?1?phosphate
CTP synthetase catalyzes the glutamine-dependent conversion of UTP to CTP. The enzyme is allosterically inhibited by the product, CTP. Mammalian cells defective in this allosteric inhibition are found to have a complex phenotype: They require thymidine in
Uncontrolled conversion of UTP to CTP elevates pools of cytidine and deoxycytidine nucleotides, while pools of uridine and thymidine nucleotide pools are diminished. The depletion of endogenous thymidine nucleotides explains the growth requirement for exo
CTP synthetase catalyzes the glutamine-dependent conversion of UTP to CTP. The enzyme is allosterically inhibited by the product, CTP. Mammalian cells defective in this allosteric inhibition are found to have a complex phenotype: They require thymidine in
The perturbed dNTP pool imbalance (dCTP/dTTP pool ratio is elevated) causes DNA replication errors, principally C incorporated opposite A, that lead to mutations.
T or F?
All six atoms in the pyrimidine ring are derived from two amino acids.
False
Mammalian cells can become resistant to the lethal action of methotrexate by the selective survival of cells containing increases in dihydrofolate reductase gene copy number so that intracellular levels of the enzyme become very high.
What other biochemic
Mutation in the DHFR structural gene rendering the enzyme resistant to MTX inhibition
induction of enzymes for MTX catabolism
mutation in gene-regulatory machinery that leads to DHFR overproduction
transport defect that blocks uptake of methotrexate into
A classic way to isolate thymidylate synthase-negative mutants of bacteria is to treat a growing culture with thymidine and trimethoprim. Most of the cells are killed, and the survivors are greatly enriched in thymidylate synthase-negative mutants.
Part A
Absence of DHF
A growth requirement of cells for thymidine
A classic way to isolate thymidylate synthase-negative mutants of bacteria is to treat a growing culture with thymidine and trimethoprim. Most of the cells are killed, and the survivors are greatly enriched in thymidylate synthase-negative mutants.
PartB
The mutants have negligible flux through the thymidylate synthase reaction because of the enzyme deficiency, they do not deplete their intracellular tetrahydrofolate coenzyme pools, which remain available for functions other than dTMP synthesis.
The added
A classic way to isolate thymidylate synthase-negative mutants of bacteria is to treat a growing culture with thymidine and trimethoprim. Most of the cells are killed, and the survivors are greatly enriched in thymidylate synthase-negative mutants.
Part C
Use methotrexate instead of trimethoprim for the selection, because trimethoprim is an ineffective inhibitor of mammalian dihydrofolate reductases.
The text states that a side effect of 5-fluorouracil therapy is its incorporation into RNA.
Part A
Choose the correct sequence of pathways by which 5-fluorouracil could be converted to FdUMP.
1) FU+deoxyribose?1?phosphate?Fluorodeoxyuridine(FUdR)+Pi (thymidine phosphorylase)
2) Fluorodeoxyuridine+ATP?F?dUMP+ADP
The text states that a side effect of 5-fluorouracil therapy is its incorporation into RNA.
Part B
Choose the correct sequence of pathways by which 5-fluorouracil could be converted to an immediate RNA precursor.
1) FU+ribose?1?phosphate?Fluorouridine+Pi (uridine phosphorylase)
2) Fluorouridine+ATP?F?UMP+ADP
3) F?UMP+ATP?F?UDP+ADP
4) F?UDP+ATP?F?UTP+ADP
Which statement about nucleotides is FALSE?
1.The only role of deoxyribonucleotides is as constituents of DNA.
2.The electrons for reduction of ribonucleotides to the corresponding deoxyribonucleotides ultimately come from NADPH.
3.dTTP can only be synthe
dTTP can only be synthesized from dCDP
T or F?
All of the dNTPs are produced in approximately the same amounts because the first step in each pathway is catalyzed by the same ribonucleotide reductase enzyme
False
T or F?
The mechanism of action of dihydrofolate reductase inhibitors used for chemotherapy is based on their ability to block the synthesis of dTMP.
True
Consider the large number of therapeutically effective nucleoside analogs.
Why do you suppose that these drugs are administered as nucleosides, rather than as nucleotides, which would be more readily converted to the active form of the drug?
Nucleotides, being charged by the presence of phosphate, can be taken up into cells only by specific transport systems, whereas uncharged nucleosides are readily taken up into most cells by facilitated diffusion.
Sulfonamide drugs like sulfanilamide inhibit tetrahydrofolate biosynthesis.
What intermediate or intermediates in purine synthesis would you expect to accumulate in sulfanilamide-treated bacteria?
glycinamide ribonucleotide and 5-aminoimidzole-4-carboxamide
Suppose that a cultured mammalian cell line was treated with sulfanilamide. What intermediates might accumulate in these cells?
Mammalian cells are not influenced by sulfanilamide, therefore no products will accumulate.
If you were to determine whether a similar reaction converts adenine directly to dAMP, what metabolite would you need to find in cells at appreciable concentrations
1. 3-Phospho-2'-deoxyribosyl-1-pyrophosphate
2 .5-Phospho-2'-deoxyribosyl-3-pyrophosphate
3. 5-Phospho-2'-deoxyribosyl-1-pyrophosphate
The purinosome contains enzymes that convert the serine hydroxymethyl group to the formyl group of 10-formyltetrahydrofolate
1)Serine+THF?Glycine+5,10?methylene?THF+H2O2) 5,10?METHYLENE-THF+NAD(P).?5,10?methenyl?THF+NAD(P)H+H+3) 5,10?methenyl?THF+H2O?10?formyl?THF
The purinosome contains enzymes that convert the serine hydroxymethyl group to the formyl group of 10-formyltetrahydrofolate
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