Nucleotides

: adenosine, adenosine deaminase : base, xanthine, urate, xanthine oxidase :

Purine Catabolism : AMP is dephosphorylated to ______ and the 6-amino group is converted to a keto group by ______. : The _____ is removed and then oxidized first to ______ and then to _____ by _____. Adenosine => Inosine + NH4+ (Adenosine deaminase) Inosine + Pi => Hypoxanthine + Ribose 1-P (Nucleoside phosphorylase) Hypoxanthine + H2O + O2 => Xanthine + H2O2 (Xanthine oxidase) Xanthine + H2O + O2 => Uric acid + H2O2 (Xanthine oxidase)

Adrucil, Efudex, Fluorplex

some drug names of 5-Fluorouracil

: diffuse away : cluster together, purinosome

Notes about Purine Synthesis : As in the case of pyrimidines, multifunctional enzymes containing several active sites catalyze many of the biosynthetic steps. : This allows the "channeling" of substrate from active-site to active site and speeds up the synthesis. : The intermediates are not released into solution where they might ______. : Furthermore, all 6 proteins ______ during active purine synthesis to optimize synthesis. (complex is termed a "_____".)

1. pyrimidine : glutamine, ammonia => cytoplasm, mitochondrial, N-Ac-Glu : glutamine, pyrimidine, antineoplastic

1. CPSII is the committed step of ______ synthesis. : This enzyme catalyzes a slightly different reaction than CPSI (urea cycle). : CPSII uses ______ as the N-donor instead of _____. => This makes sense in light of each enzyme's physiological role. CPSII is located in the _____. CPSI is ______. CPSII is not stimulated by ______. : Azaserine is a glutamine analog that inhibits many enzymes that use ______ as a N donor. The azo moiety resembles the side-chain amide of glutamine. The azo group is highly reactive and forms covalent bonds with amino acid side chains in the amide-binding pocket. Given that azaserine is an inhibitor of _____ synthesis, it has use as an ______ drug.

: CAD : three : labile intermediates can diffuse through a tunnel in the protein between active sites and are not exposed to the cruel world outside the enzyme.

2. The first three enzymes of pyrimidine synthesis are all present on the same polypeptide, termed _____ (i.e., they are encoded by a single gene). : The protein has multiple active sites that catalyze the first _____ steps of pyrimidine synthesis. : The advantage of this arrangement is that ...?

3. ATCase : ATCase

3. In bacteria, ______ is the major regulated step of pyrimidine synthesis, unlike humans. : ______ was previously encountered in this course as an example of an enzyme that is regulated by allosteric mechanisms.

4. inner mitochondrial membrane : ubiquinone : Orotate : UMP

4. Dihydroorotate dehydrogenase is located on the _______ in the intermembrane space. : Dihydroorotate diffuses through the outer mitochondrial membrane and is oxidized by the enzyme. : The electrons from dihydroorotate are passed to ______ via an enzyme-bound FMN prosthetic group and enter electron transport. : _____ then diffuses out of the mitochondria where the synthesis of _____ is finished in the cytoplasm.

: alpha : beta : uridine, cytosine, de novo synthesis and in salvage reactions

5. PRPP is the ribose-P donor for all nucleotide synthesis. : The anomeric configuration of PRPP is _____. : The N-glycosidic bonds in nucleotides are ______. : Orotate phosphoribosyl transferase will also accept ______ and cytosine as substrates and therefore functions both in ____ and _____

6. UMP : C, T : aspartate : bicarbonate, glutamine HCO3- + Gln + 2ATP +Asp + Q + PRPP => UMP + Glu + 2ADP + 2Pi + PPi + QH2 + CO2

6. Decarboxylation gives us ______. : UMP is the precursor to both ____ and ____ nucleotides. : The enzymes catalyzing steps 5 and 6 form a heterodimer. : Regarding the ring, atoms N1, C4, C5 and C6 are derived from ______. : C2 is derived from ______ and N3 comes from the side chain of _____. The overall stoichiometry is:

: sulfite :

: Mo is a cofactor for a number of enzymes including sulfite oxidase and aldehyde oxidase. : In these enzymes, the Mo tightly bound as a pterin cofactor called 'molybdopterin' or more simply, "MOCO". MOCO is synthesized from GTP and molybdate (MoO42-) we get in our diet. : A genetic deficiency that impairs MOCO synthesis leads to ______ accumulation and toxic effects beginning at birth. : In mammals other than primates, uric acid is further metabolized to allantoin by the enzyme, uric oxidase (aka uricase). => This enzyme breaks the 6-member ring of urate and releases C-6 as CO2. => Allantoin is more water-soluble than urate. => Birds also lack uricase and therefore excrete their purines as urate. => Most reptiles and certain other non-mammals catabolize allantion to urea. Fish excrete NH4+.

: purine

: Three commonly consumed stimulants, caffeine (coffee), theophylline (tea), and theobromine (chocolate), are ______ derivatives. => Theophylline lacks the methyl group on N-7 (N1,N3-dimethylxanthine). => Theobromine lacks the methyl group on N-1.

: NMPs, dephosphorylated : ATP : ATP, NDPs, NTP

: pyrimidines and purines are synthesized as ____ but can be _____ to the nucleoside. : They can then be rephosphorylated using _____, and then further phosphorylated to the triphosphate : The last two reactions are reversible so one can make ____ from two ____ or from another _____. : The high fraction of ATP in normal cells ensures that all the nucleotides exist primarily as triphosphates.

: keto, amino : aspartate : succinyl-AMP, fumarate, AMP : GTP : GTP, IMP, AMP

AMP Synthesis : To make AMP, the 6-_____ group is modified to an _____ group. : Just like the N1 nitrogen, the amino group comes from ______. : A _______ intermediate is formed and _____ is released generating ____. IMP + Asp + GTP => Adenylosuccinate + GDP + Pi => AMP + fumarate : The first step uses _____ as the energy donor. : This helps in balancing nucleotide pools- as _____ levels increase this increases the conversion of _____ to ______.

: synthesizing DNA : S : polyubiquitination : dNTPs, pyrimidine dNDPs, dGDP, dATP :

Activity of ribonucleotide reductase : The enzyme is abundant only in cells that are actively _______. : Transcription of R2 is dramatically induced during the _____ phase in actively dividing cells. : During mitosis, R2 is actively degraded by ______, once dNTPs are no longer needed. : Tight control is also maintained by allosteric mechanisms; high levels of _____ allosterically inhibit this enzyme (feedback inhibition). However, it is critical that a balance of dNTPs be present, since absence of a single dNTP will bring DNA synthesis to a crashing halt. Thus, dATP and ATP stimulate the formation of _________, whereas TTP stimulates _______ synthesis and dGTP stimulates ______ synthesis. : Positive allosteric modulation by the other dNTPs keeps all 4 dNTPs at approximately equal concentrations.

: gout : xanthine oxidase, urate : suicide inhibitor => alloxanthine

Allopurinol and Febuxostat : Allopurinol can be used to prevent ______. : It works by inhibiting _______ and therefore prevents the accumulation of ______. : A new agent, Febuxostat (Uloric) also inhibits xanthine oxidase. : Allopurinol is a ______. => Allopurinol is oxidized by XO to ______. => Alloxanthine is trapped at the enzyme active site and the enzyme cannot complete the catalytic cycle. (Xanthine oxidase has a Mo cofactor at the active to aid in transferring electrons. The Mo cycles between the Mo(IV) and the Mo(VI) state. Alloxanthine traps the Mo at the 4+ state).

1. PRPP, PRPP synthetase, purine or pyrimidine synthesis. 2. PRPP Amidotransferase => N-glycosidic => glutamine 3. Glycine => ATP 4. N10-formyl-tetrahydrofolate 5. glutamine, ATP for energy. 6. ATP for energy 7. biotin nor ATP 8. aspartate, glutamine, succinate, fumarate 9. N10-formyltetrahydrofolate 10. inosine monophosphate (IMP)

De Novo Purine Synthesis 1. The first step is the synthesis of _____ catalyzed by ________. The PRPP made is used for either ____ or _____ 2. __________________ => This is the committed step of de novo purine synthesis. In this reaction, the _______ bond is formed. => As is common in anabolic reactions, the side chain nitrogen of ______ is the N donor. PRPP + Gln + H2O => 5' phosphoribosylamine + Glu + PPi 3. ______ is added to 5'-phosphoribosylamine and becomes C4, C5, and N7 of the purine ring. => Reaction requires ______ to form the amide bond. 4. The final atom of the 5 member ring, C8, is added from _______. 5. The N3 atom of the 6 member ring is added from ______. Requires _______ 6. The 5 member ring is closed. Requires ______ 7. CO2 is added to provide the C-6 atom. Unusual in that neither ____ or _____ is needed. 8a, b. In a reaction very similar to the sequence in the urea cycle, N1 is added from ______ (not _____) to make the ______ derivate. Then ______ is cleaved away. 9. The final atom of the purine, C2 is added from _________. 10. The 6 member ring is closed to form ____________.

: assembled while attached to the ribose 5-P. (from PRPP) : folate, bicarbonate : inosine monophosphate (IMP), IMP, AMP, GMP

De novo Synthesis : Unlike pyrimidines the base is ...?? : The synthesis of PRPP is described above and is an important regulatory step. : The atoms that make up the purine ring are added in a series of steps from amino acid precursors, _____ cofactors and a ______ (from CO2). : The first purine made is _______. From _____, the synthesis diverges to produce either _____ or _____.

: diphosphate : ribonucleotide reductase, free radical : R1, R2, tyrosine, Cys : C2-OH : Cys, disulfide : thioredoxin, Thioredoxin : a flavoprotein, thioredoxin reductase, glutathione

Formation of deoxynucleotides : Deoxynucleotide formation takes place at the ______ level. : This reaction is catalyzed by the enzyme _______, and involves a ______ mechanism. : Ribonucleotide reductase contains two dissimilar subunits, _____ and _____. A ______ free radical is produced in the R2 subunit and the free radical is transmitted to a ____ in the active site of the R1 subunit. : The _____ is reduced to H and the OH released as H2O. : During the reaction, two ____ residues are converted to a _____. : To refresh the enzyme for another substrate, ribonucleotide reductase is reduced by a small protein cofactor, _______. _______ is a member of a family of small redox active proteins involved in reducing oxidized proteins, [whether oxidized as a normal part of metabolism or by undesirable ROS]. : Thioredoxin itself is regenerated by ______, which uses electrons from NADPH like all good anabolic reactions. Thioredoxin reductase is related to _____ reductase

TDP = thiamine diphosphate : dUMP

Formation of deoxynucleotides _____ does not exist and is not a substrate for this reaction. : T is formed from _____ after the formation of the deoxynucleotide

: keto, amino : xanthosine monophosphate (base is xanthine) : glutamine : ATP, ATP, GMP

GMP Synthesis : The conversion of IMP to GMP is a 2 step conversion. : The C-2 carbon is oxidized to a _____ group and then the keto group is converted to an _____ group. : The intermediate nucleotide is ______. IMP + NAD+ + H2O => XMP + NADH XMP + Gln +ATP => GMP + Glu + AMP + PPi : This time _____ is the N donor. : ____ provides the energy. Again, using ____ ensures the appropriate nucleotide balance; ie, as ATP levels go up, it increases the conversion of IMP to _____.

: hypoxanthine : inosine : inosinic acid : folate : T, C or U, THF, DHF, DHFR

IMP is the first purine product : To make AMP and GMP the synthesis diverges. : IMP is an important intermediate point in the synthesis. : Because the synthesis diverges, utilization of IMP is another important regulatory point. Terminology: : The base portion of IMP is termed _______. : The nucleoside is ______. : The nucleotide is ______.(IMP) The stoichiometry to this point is: PRPP + 2Gln + Gly + CO2 + Asp + 3ATP + 2 N10-formyl-THF => IMP + 2Glu + 2 THF + fumarate + H20 + 2 ADP + 2Pi + PPi : Note that: _______ cofactors are required at two steps in ring formation. : Contrast this with pyrimidines where folate is only required for _____, not _____. (The cofactor product is ______, not ______ as in T synthesis- no need for ______ here)

: hydrogen peroxide : severe combined immunodeficiency => dATP, dNTPs => Lymphocytes

Purine Catabolism : Notice _____ is generated by xanthine oxidase. Ouch! Good thing urate is an antioxidant. : A rare genetic deficiency in adenosine deaminase (ADA) gives rise to _______. => As a result, ______ levels rise and inhibit the production of other ______. => ______ are unable to proliferate and mount an immune response. => This was one of the first genetic diseases for which gene therapy was tried.

: uric acid/ urate => antioxidant : soluble => precipitate out

Purine Catabolism : The breakdown of purines is clinically significant. : Purines are metabolized to ______. : Urate is then excreted in the urine. => This molecule can act as an ______. : Of clinical relevance is the fact that urate is only weakly_____. => When urate accumulates, especially in the acidic environment of the urine, it can ______ and cause difficulties. => In the body, gout results from the accumulation of insoluble urate crystals in tissues. => When the crystals accumulate in joints it can lead to painful bone erosions and inflammation. Apparently, even the mighty T. rex suffered from gout. [Nature 387:357 (1997)]

: xanthine : guanosine, base : xanthine

Purine Catabolism : The catabolism of GMP converges with that of AMP at _____. : GMP is dephosphorylated to ______, which is phosphorylyzed to release the ______. : The C-2 amino group is then deaminated leading to _____. Guanosine + Pi => Guanine + Ribose 1-P (Nucleoside phosphorylase) Guanine + H2O => Xanthine + NH4+ (Guanase)

: PRPP : 5'P-ribosylamine : AMP, GMP and IMP => PRPP is required for other pathways : PRPP amidotransferase => AMP, GMP and IMP => AMP => GMP

Purine Synthesis Regulation : The flux through the pathway is controlled primarily by the availability of ______. : As PRPP increases, more ______ is made. : ________ are negative allosteric modulators of PRPP synthetase. => They do not totally inhibit the enzyme because ...? : The committed step of purine synthesis is ________ (#2 above). => This step is also feedback inhibited by ______ : Branch points => _____ inhibits its own synthesis from IMP while GTP is stimulatory. => _____ inhibits its own synthesis from IMP whereas ATP is stimulatory. => This branch point regulation helps insure a balanced pool of nucleotides.

: amino acids : purine, PRPP, 5- OPRT

Purine synthesis : We distinguish two types of purine nucleotide synthesis, de novo and salvage. : In de novo synthesis, the purines are synthesized from scratch, from _______ and derivatives, as we described for pyrimidines. : In salvage pathways, the ______ base is recycled. => After being removed from the nucleotide it is reattached to ______. (The salvage pathway of pyrimidine synthesis coincides with de novo synthesis at enzyme _______).

: water, ß-alanine, ß-aminoisobutyric acid : Coenzyme A-CoASH, succinyl CoA , acetyl CoA

Pyrimidine Catabolism : The pyrimidines are broken down to ______-soluble catabolites: ________ and ___________. : These may be excreted in the urine, used for the synthesis of other compounds (ß-alanine is part of __________), or further catabolized to ___________ and __________. : Nucleotides, however, are not present in high enough concentrations to be a significant source of calories. (The pathway is shown in the slides)

: bicarbonate, glutamine and aspartate : pyrimidine, ribose-P, ribose-P : CMP, UMP

Pyrimidine Synthesis Overview : The precursors of the pyrimidine ring are ____. : These molecules come together to form a _____ ring that is then added to the ______ (this is in contrast to purine synthesis in which the purine is built on the ______). : Finally, _____ is made directly from _____.

: phosphorylation : UTP : PRPP : ATP, ATP : MAP kinase, CPSII, UTP : CAD

Regulation of Pyrimidine Synthesis : The major regulated step is catalyzed by CPSII. : CPSII is subject to both allosteric regulation and regulation by ______. : ______ is a feedback inhibitor of CPSII. : ______ is an activator of CPSII. : High levels of _____ stimulate pyrimidine synthesis (and therefore maintain balanced nucleotide pools) because _____ is a substrate for CPSII. : In actively dividing cells, the need for DNA precursors is increased. To meet this need. CPSII is phosphorylated by ______, which increases ______ activity and prevents feedback inhibition by _____. : In addition, transcription of the _____ gene is elevated in actively dividing cells. (MAP kinase effect)

: glutamine amidotransferase : irreversible : both pyrimidine and purine synthesis.

Review : Azaserine is a glutamine analog that inhibits ______ reactions (steps 2 and 5). : The azo moiety reacts with nucleophilic side chains in the enzymes leading to covalent modification and ______ inhibition. : Azaserine inhibits ...?

: phosphorolysis, ribose 1-P + base : base : adenine phosphoribosyl transferase (APRT => PRPP

Salvage Pathways (Purine Synthesis) : Purine bases can be salvaged and recycled. This is apparently quite important in the brain where nucleosides can function as neuromodulators. : Their action is terminated by ______ of the nucleoside to _______. : The _____ is then recycled. : Adenine is recycled by the enzyme _____. => _______ is the ribose-P donor. Adenine + PRPP => AMP + PPi

: IMP, GMP, hypoxanthine-guanine phosphoribosyl transferase (HPRT) : APRT : HPRT => urate => purines => brain => PRPP, HPRT => de novo => purines, urate

Salvage Pathways (Purine Synthesis)- Hypoxanthine/Guanine : Hypoxanthine can be recycled to ______ and guanine can be recycled to ______ by the enzyme _______. : The reaction is the same as that for _______. : The genetic disease Lesch-Nyhan syndrome is due to the absence of ______. => The gene is located on the X chromosome and the disease is recessive. => It is associated with elevated _____ in the urine, which can show up as crystals in the boy's diapers. => The syndrome leads to mental retardation, spasticity, gout and bizarre behavior such as self-mutilation. => The liver makes excess ______ for other tissues. => The ______ is reliant on this exogenous source of purines. => The disease is compounded by the fact that _____ increases since it is not being used by _____. => The excess PRPP feeds into the _____ pathway and elevated ______ and _______ result.

: glutamine, keto, amino, triphosphate (UTP) : GTP, CTP : dephosphorylate CTP to CDP, deoxy to dCDP, rekinase to dCTP

Synthesis of C : This occurs in a single-step reaction in which the side chain N of ______ is used to convert the 4-_____ group into a 4 _____ group. This takes place at the _____ level. UTP + Gln + ATP => CTP + Glu + ADP + Pi (CTP synthetase) : This reaction is stimulated by _____ and inhibited by _____ - think balanced pools. (Note: to make dCTP you first have to ________, then _________ and _________.)

: monophosphate, dUMP, UMP : methylation : N5, N10-methylene tetrahydrofolate : methylene, electrons, dihydrofolate : U is not incorporated into DNA : dividing cells : anticancer agents and antibiotics.

Synthesis of T : The T nucleotide is made at the _______ level from ______ (NOT ______). : The synthesis of TMP is a reductive _______. : The required cofactor is ________. : The cofactor donates both a ______ group and 2 ______ and the THF is oxidized to ________. dUMP + N5,N10-methylenetetrahydrofolate => TMP + dihydrofolate (thymidylate synthase) : Thymidylate synthase localizes to the nucleus, which helps insure that ________. : Given that T is only found in DNA, it follows that thymidylate synthase is highly active only in _______. : Thus, drugs that affect thymidylate synthase should make effective ____ and _____

: phosphoribosylpyrophosphate (PRPP) : purine and pyrimidine nucleotides

Synthesis of the ribose donor : The ribose is "activated" for glycosidic bond formation by conversion to ________. : Ribose 5'-phosphate comes from pentose-P pathway. : Regulation: PRPP synthetase is subject to feedback inhibition by both _____

1st

The Pyrimidine Pathway : what is major regulated step? : Note where phosphoribosylpyrophosphate (PRPP) comes in