DNA: Nucleotide Metabolism Flashcards
Universal energy currency in all cells. ____ also used.
ATP. GTP.
Participates in many biochemical reactions
Purines & Pyrimidines
3 major coenzymes
NAD+, FAD & Coenzyme A
Are components of 3 major coenzymes
Adenine Nucleotides
Are activated intermediates in many biosynthesis
Nucleotide derivatives
Biosynthesis: UDP to
Glucose to Glycogen
Biosynthesis: CDP to
Diacyglycerol to Phosphoglycerine
Are metabolic regulators
Nucleotides
2nd messenger in many hormone actions
cAMP
Are activated precursor in nucleic acid synthesis.
Nucleoside triphosphates
Critical reactions in ______ & ______ biosynthesis are target for cancer chemotherapy and viral disease treatment.
Purine & Pyrimidine.
In purine nucleotide synthesis, Purine Ribonucleotides formed ________. Purines are not initially synthesized as ______.
De novo. Free bases.
First purine derivative is
Inosine Mono-phosphate(IMP)
AMP & GMP are formed from
IMP
Enumerate the 10 biosynthetic steps in purine biosynthesis
“PAFNR CAFFR” *refer to handout
1st step in Purine Biosynthesis: ___ displacement by ___. Thru
PP. NH2. Glutamine.
2nd step in Purine Biosynthesis: addition of ________. Thru
Glycine. ATP + Glycine
3rd step in Purine Biosynthesis: ______ group transfer. Thru
Formyl. N10Formyl THF
4th step in Purine Biosynthesis: ___ transfer. Thru
NH2. ATP + Glutamine
5th step in Purine Biosynthesis: _____ closure. Thru
Ring. ATP.
6th step in Purine Biosynthesis: ______ addition. Thru
COO-. CO2
7th step in Purine Biosynthesis: _____ addition. Thru
Aspartate. ATP + Aspartate
8th step in Purine Biosynthesis: ________ loss. Thru
Fumarate. Fumarate
9th step in Purine Biosynthesis: _______ group transfer. Thru
Formyl. N10Formyl THF
10th step in Purine Biosynthesis: ring _______.
Closure
The activated sugar used in purine nucleotide synthesis is
5-phosphoribosyl-1-pyrophosphate “PRPP”
PRPP is generated by the action of __________ and requires energy in the form of ____. This action releases ____. Therefore , 2 high energy ______ equivalents are consumed during the reaction. First committed step in purine synthesis (regulated) Inhibited by ____ & ____ (allosteric)
PRPP synthetase. ATP. AMP. Phosphate. ADP & GDP.
The major site of purine synthesis
Liver
Synthesis of purine nucleotides begin with _____ and leads to the first fully formed nucleotide, ___________.
PRPP. Inosine 5’-monophosphate “IMP”
The purine base is built upon the ribose by several _________ & _______ reactions.
Amidotransferase. Transformylation.
The synthesis of IMP requires __ moles of ATP, __ moles of glutamine, __ mole of glycine, __ mole of CO2, __ mole of aspartate and __ moles of formate.
- 2.
The formyl moieties are carried on tetrahydrofolate (THF) in the form of ____________ and ___________.
N5, N10. Methenyl- THF and N10-formyl-THF.
Sources of atom in Purine Biosynthesis
Glycine, Glutamine, Aspartate, N5 N10 Methenyl THF, N10 Formyl THF & CO2. “GGANNC”
Synthesis of AMP/GMP: ___ represents a branch point of purine biosynthesis, because it can be converted into either ___ or ___ through two distinct reaction pathways.
IMP. AMP or GMP.
Synthesis of AMP/GMP: The pathway leading to AMP requires energy in the form of ___; that leading to ___ requires energy in the form of ___.
GTP. GMP. ATP.
Synthesis of AMP/GMP: allows the cell to control the proportions of AMP & GMP to near equivalence.
Utilization of GTP in the pathway to AMP
Synthesis of AMP/GMP: the accumulation of excess GTP will lead to accelerated ___ synthesis from ____ instead., at the expense of ___ synthesis.
AMP. IMP. GMP.
Synthesis of AMP/GMP: Conversely, since the conversion of IMP to GMP requires ATP, the accumulation of excess ATP leads to accelerated synthesis of ___ over that of ___.
GMP. AMP.
The essential rate limiting steps in purine biosynthesis occur at the
First two steps of the pathway
The synthesis of PRPP by PRPP synthetase is feed-back inhibited by
Purine-5’-nucleotides. (Predominantly AMP & GMP)
The amidotransferase reaction (2nd step) catalyzed by _________ is also feedback inhibited by binding ATP, ADP & AMP at one inhibitory site and GTP, GDP & GMP at another. Conversely the activity of the enzyme is stimulated by _____.
PRPP glutamyl amidotransferase. PRPP.
Purine biosynthesis is regulated in the branch pathways from IMP to AMP and GMP. The accumulation of excess AMP leads to accelerated synthesis of ___,and excess of this leads to accelerated synthesis of ___.
GMP. AMP.
Catabolism of the purine nucleotides leads ultimately to the production of _______ which is insoluble and is excreted in the urine as _________.
Uric acid. Sodium urate crystals.
The synthesis of nucleotides from the purine bases and purine nucleosides takes place in a series of steps known as the
Salvage pathways
The free purine bases _______, ______ & ______, can be converted to their corresponding nucleotides by ___________.
Adenine. Guanine. Hypoxanthine. Phosphoribosylation.
Two key transferase enzymes that are involved in the salvage of purines
Adenosine phosphoribosyltransferase(APRT) and Hypoxanthine-guanine phosphoribosyltransferase(HGPRT)
Hypoxanthine + PRPP IMP + PPi and Guanine + PRPP GMP + PPI. Catalyzed by
Hypoxanthine-guanine phosphoribosyltransferase(HGPRT)
Adenine + PRPP AMP + PPI. Catalyzed by
Adenosine Phosphoribosyltransferase
A critically important enzyme of purine salvage in rapidly dividing cells which catalyzes the deamination of adenosine to inosine.
Adenosine deaminase
Deficiency in ADA results in the disorder called
Severe combined immunodeficiency “SCID”
Can also contribute to the salvage of the bases through a reversal of the catabolism pathways. However, this pathway is less significant than those catalyzed by the phosphoribosyltransferases.
Purine nucleotide phosphorylases
The synthesis of AMP from IMP and the salvage of IMP via AMP catabolism have the net effect of deaminating _______ to _______. This process has been termed as
Aspartate. Fumarate. Purine nucleotide cycle.
This cycle is very important in muscle cells, because increases in the muscle activity create a demand for an increase in the TCA cycle, inorder to generate more ____ for the production of ___.
Purine nucleotide cycle. NADH. ATP.
However, muscle lacks most of the enzymes of the major ______ reactions. Muscle thus replenishes TCA cycle intermediates in the form of _______ generated by the purine nucleotide cycle.
Anaplerotic. Fumarate.
Is the muscle-specific isoenzyme of AMP deaminase, and deficiencies in this leads to post-exercise fatigue, cramping and myalgias.
Myoadenylate deaminase
Separate kinase for each nucleotide
Nucleoside monophosphate kinases
Single enzyme with broad specificity
Nucleoside Diphosphate Kinase
Is a condition that results from the precipitation of urate (uric acid) as monosodium urate (MSU) or ___________ crystals in the synovial fluid of the joints, leading to severe inflammation and arthritis.
Gout. calcium pyrophosphate dihydrate
Most forms of gout are the result of excess _______ production or to a partial deficiency in the salvage enzyme, ______.
Purine. HGPRT.
Example for gout, increased activity of PRPP synthetase leads to excess PRPP leading to increased _________ production that can increase the rate of purine degradation and subsequently increases ________ synthesis.
Purine nucleotide. Uric acid synthesis.
Responsible for reforming IMP & GMP from hypoxanthine and guanine
HGPRT & salvage pathway
Most forms of gout can be treated by administering this antimetabolite. This compound is a structural analog of hypoxanthine that strongly inhibits _________.
Allopurinol. Xanthine oxidase.
Results from the loss of a functional HGPRT gene. The disorder is inherited as a sex-linked trait, with the HGPRT gene on the X chromosome. Patients with this defect exhibit not only symptoms of gout but also a severe malfunction of the nervous system. Patient may result to self-mutilation. Death before 20th year.
Lesch-Nyhan syndrome
Most often caused by a deficiency in the enzyme adenosine deaminase. ADA deficiency selectively leads to destruction of ________, the cells that mount immune system.
Severe Combined Immunideficiency disease (SCID). B & T lymphocytes.
In the absence of ADA, ______ is phosphorylated to yield levels of dATP that are 50-fold higher than normal. The levels are especially high in _______, which have abundant amounts of the salvage enzymes.
Deoxyadenosine. Lymphocytes.
High concentration of dATP inhibit _________ thereby preventing other dNTPs from being produced. The net effect is to inhibit ________.
Ribonucleotide reductase. DNA synthesis.
Since lymphocytes must be able to proliferate dramatically in response to antigenic challenge, the inability to synthesize DNA seriously impairs the ________, and the diseases is usually fatal unless special protective measures are taken.
Immune response
A less sever immunodeficiency results when there is lack of ________, another purine degradative enzyme.
Purine nucleoside phosphorylase.
A glycogen storage disease which leads to excessive uric acid production. This disorder results from a deficiency in ______ activity.
Von Gierke’s disease. Glucose 6 Phosphatase
The increased availability of ____ increases the rate of flux through the PPP, yielding an elevation in the level of ____ and consequently _____ then result in excess _________.
G6P. R5P. PRPP. Purine biosynthesis.
Elevated PRPP synthetase and deficiency in HGPRT & G6P
Gout
HGPRT lacking
Lesch-Nyhan syndrome
ADA lacking
SCID
PNP lacking
Immunideficiency
APRT lacking
Renal lithiasis
Xanthine oxidase absent
Xanthinuria
G6P deficiency
von Gierke disease
Synthesis of ______ is less complex than that of purine, since the base is much simplier. The first completed base is derived from __ mole of glutamine, __ mole of ATP, __ mole of CO2 and __ mole of aspartate.
Pyrimidines. 1. 1. 1. 1.
Carbamoyl phosphate used for pyrimidine nucleotide synthesis is derived from _______ & _______, within the _______. As opposed to the urea cycle CP derived from ______ & _______ in the _______.
Glutamine & Bicarbonate. Cytosol. Ammonia & Bicarbonate. Mitochondrion.
The pyrimidine nucleotide precursor is synthesized by
Carbamoyl Phosphate Synthetase II
Carbamoyl phosphate is the condensed with aspartate in a reaction catalyzed by the rate limiting enzyme of pyrimidine nucleotide biosynthesis known as
Aspartate Transcarbamoylase (ATCase)
UMP synthesis overview: 2 ATPs needed both used in first step. One transfers _____, the other is hydrolyzed to _____. 2 condensation reactions: form _________ and ___.
Phosphate. ADP & Pi. Carbamoyl Aspartate & OMP.
Pyrimidine ring is synthesized from
Carbamoyl phosphate and Aspartate
Carbamoyl P for pyrimidine synthesis is formed in _____. Enzyme is ___________. And uses ________ as an N-donor.
Cytosol. CP II. Glutamine.
Are attached to PRPP AFTER synthesis
Pyrimidines
Diff. in Purine & Pyrimidine Biosynthesis: 3. The ring structure is assembled as a _______, not built upon PRPP. PRPP is added to the first fully formed pyrimidine base (AKA _______) forming ______ which is subsequently decarboxylated to UMP.
Pyrimidine base. Orotic acid. Orotate Monophosphate (OMP)
Diff. in Purine & Pyrimidine Biosynthesis: 2. there is no branch in the pyrimidine synthesis pathway. UMP is phosphorylated twice to yield ____. (ATP is a phosphate donor). The first phosphorylation is catalyzed by ________ and then second by __________.
UTP. Uridylate kinase. Ubiquitous nucleoside diphosphate kinase.
Diff. in Purine & Pyrimidine Biosynthesis: 3. UTP is aminated by the action of _________, generating CTP. The thymine nucleotides are in turn derived by _____ synthesis from dUMP or by salvage pathways from _______ or _______.
CTP synthase. De novo. Deoxyuridine or Deoxythymidine.
Deoxyribonucleotide formation: purine/pyrimidine degradation are the same for ribonucleotides and deoxyribonucleotides. Biosynthetic pathways are only for ________ production. _________ are synthesized from corresponding ribonucleotides.
Ribonucleotide. Deoxyribonucleotide.
DNA composed of _________. Ribose sugars in DNA lacks hydroxyl group at ______. ______ doesn’t appear in DNA (normally), ______ appears instead.
Deoxyribonucleotides. 2’ carbon. Uracil. Thymine (5-methyluracil)
Formation of Deoxyribonucleotides: reduction of 2’ carbon done via a ________ catalyzed by __________.
Free radical mechanism. Ribonucleotide Reductases.
Is a multifunctional enzyme that contains redox-active thiol groups for the transfer of electrons during reduction reactions. In the process of reducing _____ to _____, this becomes oxidized.
Ribonucleotide reductase. rNDP. dNDP.
RR is reduced in turn by either _______ or _______. The ultimate source of the electrons is ______.
Thioredoxin or Glutaredoxin. NADPH.
The electrons are shuttled through a complex series of steps involving enzymes that regenerate the reduced forms of thioredoxin and glutaredoxin. These enzymes are
thioredoxin reductase and glutathione reductase
In RR: Activates the reduction CDP & UDP
ATP
In RR: induces GDP reduction and inhibits reduction of CDP & UDP.
dTTP
In RR: inhibits reduction of all nucleotides
dATP
In RR: stimulates ADP reduction. Inhibits CDP, UDP, GDP reduction
dGTP
Synthesis of Thymine Nucleotides: the de novo pathway to dTTP synthesis first requires the use of ____ from the metabolism of either ____ or ____. The dUMP is converted to dTMP by the action of _______.
dUMP. UDP or CDP. Thymidylate synthase.
Synthesis of dTMP from dUMP: 1. the methyl group (thymine is 5-methyl uracil) is donated by _________, similarly to the donation of methyl groups during the biosynthesis of purines.
N5,N10-methylene tetrahydrofolate
Synthesis of dTMP from dUMP: 2. the unique property of the action __________ is that the THF is converted to _________. The only such reaction yielding DHF & THF.
Thymidylate synthase. Dihydrofolate.
Synthesis of dTMP from dUMP: 3. in order for the thymidylate synthesis to continue, THF must be degenerated from DHF. This is accomplished through the action of ___________.
Dihydrofolate reductase (DHFR)
Synthesis of dTMP from dUMP: 4. THF is them converted to N5, N10-THF via the action of __________.
Serine hydroxymethyl transferase
Synthesis of dTMP from dUMP: 5. The crucial role of ____ in thymidine nucleotide biosynthesis makes it an ideal target for chemotherapeutic agents such ______ & ______, which are structural analogues of _____ & ______.
DHFR. Sulfonamides & Trimethoprim. PABA & Folic acid.
Sulfonamide susceptible organisms, unlike mammals, cannot use exogenous folate but must synthesize it from _______. This pathways is thus essential for production of ______ & ______ synthesis in bacteria.
PABA. Purine & Nucleic acid.
Mammalian cells lack the enzymes required for folate synthesis from PABA and depend on exogenous sources of folate; therefore they are not susceptible to _______.
Sulfonamides
Is also useful as an antibacterial; it does not affect mammalian cells because 50k less efficient in inhibition of mammalian ________.
Trimethoprim. Dihydrofolate reductase.
This is another DHFR inhibitor which is used in cancer chemotherapy.
Methotrexate
The salvage pathway to dTTP synthesis involves this enzyme which can use either thymidine or deoxyuridine as substrate. Thymidine+ATPTMP+ADP. Deoxyuridine+ATPdUMP+ADP.
Thymidine kinase
The activity of this is unique in that it fluctuates with the cell cycle, rising to peak activity during the phase of DNA synthesis; it is inhibited by ____.
Thymidine kinase. dTTP.
The regulation of pyrimidine synthesis occurs mainly at the first step which is catalyzed by
Aspartate transcarbamoylase, ATCase
ATCase is inhibited by
dUTP, UDP, CTP & UTP “DUCU”
ATCase is activated by
ATP
Is a multifunctional protein in mammalian cells. It is capable of catalyzing the formation of carbamoyl phosphate, carbamoyl aspartate, and dihydroorotate.
ATCase
ATCase, and therefore the activity of CPS II, is localized to the _______ and prefers _______ as a substrate.
Cytoplasm. Glutamine.
The role of ______ in ATCase regulation is to act as a comoetitive inhibitor of the glutamine binding site.
Glycine
As in the regulation of purine synthesis, ___ levels also regulate pyrimidine biosynthesis at the level of PRPP formation. An increase in the level of _____ results in an activation of pyrimidine synthesis.
ATP. PRPP.
This enzyme is competitively inhibited by UMP and to a lesser degree by CMP.
OMP decarboxylase
Is feedback inhibited by CTP and activated by GTP
CTP synthase
Catabolism of the pyrimidine nucleotides leads ultimately to ______ or ______ and NH3 & CO2.
B-alanine. B-aminoisobutyrate.
Serve as a NH2 donors in transamination of a-ketoglutarate to glutamate. A subsequent reaction converts the products to ______ or ________.
B-alanine & B-aminoisobutyrate. Malonyl CoA. Methylmalonyl CoA.
The salvage of pyrimidine bases has less significance than that of the _____, owing to the solubility of the by products of pyrimidine catabolism.
Purines
Uracil can be salvaged to form UMP through the concerted action of
Uridine phosphorylase & Uridine kinase
Formed when CMP and UDP are degraded
B-alanine
Formed when dTMP is degraded
B-aminoisobutyrate
Can be diverted to fatty acid synthesis
Malonyl CoA
Converted to succinyl CoA and can be shunted to TCA cycle
Methylmalonyl CoA
Formation of dTMP, by salvage of thymine requires
Thymine phosphorylase and Thymine kinase
The salvage of deoxycytidine is catalyzed by
Deoxycytidine kinase
Two inherited disorders affecting pyrimidine biosynthesis are the result of deficiencies in the bifunctional enzyme catalyzing the last two steps of UMP synthesis
Orotate phosphoribosyl transferase & OMP decarboxylase
Deficiency in pyrimidine catabolism result in this that causes retarded growth and severe anemia caused by hypochromic erythrocytes and megaloblastic bone marrow. _______ is also common with this.
Orotic aciduria. Leukopenia.
Orotic aciduria can be treated with these which leads to increased UMP production via the action of _________.
Uridine/Cytidine. Nucleoside kinase.
Inhibits CPS II thus attenuating orotic acid production.
UMP
Can also cause orotic aciduria because it can act as an alternative substrate and compete with orotic acid for degradation.
Allopurinol
