Metabolizam nukleotida dr Milan Nikolić

Transcription

1 Metabolizam nukleotida dr Milan Nikolić (školska )

2 Chapter 23: Nucleotide metabolism Ispitna pitanja: Biosinteza purinskih ribonukleotida Biosinteza pirimidinskih ribonukleotida Formiranje dezoksiribonukleotida Principi degradacije nukleotida

3 Biological functions of nucleotides Building blocks of nucleic acids (DNA and RNA) (genetic material and non-protein enzymes) Currency in energy metabolism (e.g. ATP, GTP) Carriers of activated metabolites for biosynthesis (e.g. UDP-glucose, S-adenosylmethionine) Components of central metabolic coenzymes (e.g. NAD+, NADP+, FAD, FMN, and CoA) Metabolic regulators and signal molecules (e.g. camp, cgmp) NB: Do not provide a significant source of metabolic energy!

4 Nomenclature of nucleic bases, nucleosides, and nucleotides

5 Two biosynthetic routes for nucleotides Non-essential nutrients, because they can be synthesized in the body 1. De novo pathways Almost all cell types have the ability to synthesize purine and pyrimidine nucleotides from low molecular weight precursors in sufficient amounts. The de novo pathways are almost identical in all organisms! 2. Salvage pathways Most organisms have the ability to synthesize nucleotides from nucleosides or bases that become available through the diet or from degradation of nucleic acids. In animals, the extra cellular hydrolysis of ingested nucleic acids represents the major route by which bases become available!

6 Reutilization and catabolism of nucleotides endonucleases: pancreatic RNAse pancreatic DNAse phosphodiesterases: usually non-specific blue-catabolism red-salvage pathways

7 PRPP: a central metabolite in both (de novo and salvage) pathways (from PPP) Enzyme (ribose-5-phosphate pyrophosphokinase) is an allosteric enzyme. Its activity is inhibited when the cell contains adequate amounts of the nucleotides (e.g. AMP, IMP and GMP). Roles of PRPP: His and Trp biosynthesis, purine salvage pathways, de novo synthesis of nucleotides.

8 1. De novo biosynthesis of purines (low molecular weight precursors of the purine ring atoms) (N10-Formyltetrahydrofolate) (N10-Formyltetrahydrofolate) Stage 1: Formation of IMP (complex process) Stage 2: Conversion of IMP to either AMP or GMP John Buchanan and G. Robert Greenberg (

9 FH 4 (or THF) Folic acid or folate (vitamin B9)

10 Synthesis of Inosine 5'-Monophosphate (IMP) Basic pathway for biosynthesis of purine ribonucleotides Starts from ribose-5-phosphate (R5P) Requires 11 steps overall Occurs primarily in the liver The metabolic pathway for the de novo biosynthesis of IMP

11 OH Step 1: Activation of ribose-5-phosphate 1 ATP AMP Committed step Ribose phosphate pyrophosphokinase α Step 2: Acquisition of purine atom N9 Gln:PRPP amidotransferase Steps 1 and 2 are tightly regulated by feedback inhibition

12 Step 3: Acquisition of purine atoms C4, C5, and N7 Step 4: Acquisition of purine atom C8 Glycinamide synthetase GAR transformylase

13 Step 5: Acquisition of purine atom N3 Step 6: Closing of the imidazole ring FGAM synthetase AIR synthetase

14 Step 7: Acquisition of C6 Step 8: Acquisition of N1 AIR carboxylase SAICAR synthetase Carboxyaminoimidazole ribonucleotide (CAIR)

15 Step 9: Elimination of fumarate Step 10: Acquisition of C2 Adenylosuccinate lyase AICAR transformylase The donation of an amino group by adding an aspartate and removing a fumarate is one of the two ways in which amino/amide groups are transferred.

16 Step 11: Ring closure to form IMP IMP cyclohydrolase Once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells) Many multifunctional enzymes: Reactions: and 7-8 and

17 Synthesis of AMP and GMP from IMP Note: GTP used to make AMP, ATP used to make GMP. C6 NB: Gln donating its amide group is the second method in which amino/amide groups are transferred.

18 Pathways from AMP/GMP to ATP/GTP Conversion to diphosphate by base-specific monophosphate kinases: GMP + ATP <---> GDP + ADP AMP + ATP <---> 2 ADP Guanylate kinase Adenylate kinase Conversion to triphosphate by nucleoside diphosphate kinase (NDK): GDP + ATP <---> GTP + ADP ΔG o 0 (ping pong reaction mechanism with phospho-his intermediate) NDK also works with pyrimidine nucleotides and is driven by mass action ATP is synthesized from ADP and P i via oxidative/photo phosphorylation or substrate level phosphorylation

19 Control of de novo purine biosynthesis (Feedback inhibition & feedforward activation) (a) Meet the need of the body, without wasting (b) AMP and GMP control their respective synthesis from IMP (feedback mechanism) Purine nucleotide biosynthesis is an energy expensive pathway and as such it is tightly regulated. PRPP activates amidotransferase ADP and GDP inhibit PRPP synthetase AMP inhibits conversion of IMP to GMP and GMP inhibits conversion of IMP to GMP ATP stimulates conversion of IMP to GMP and GTP stimulates conversion of IMP to AMP Ensured a balanced synthesis of both families of purine nucleotides!

20 De novo pyrimidine biosynthesis (much more straight forward process) Shorter pathway than for purines (6 steps) Pyrimidine ring is made first (orotic acid), then attached to ribose-p (unlike purine biosynthesis) Only 2 precursors (Asp and Gln, plus HCO 3- ) contribute to the 6-membered ring The product is UMP (uridine monophosphate) CTP and UTP are derived from UMP

21 The first 3 enzymes of the pathway are contained on a single multifunctional protein in the cytoplasm

22 Step 1: Synthesis of carbamoyl phosphate Carbamoyl phosphate synthetase (CPS) exists in 2 types: CPS-I, a mitochondrial enzyme, is dedicated to the urea cycle and Arg biosynthesis CPS-II, a cytosolic enzyme, used here; It is the committed step in animals Step 2: Synthesis of carbamoyl aspartate activated compound ATCase: aspartate transcarbamoylase

23 Step 3: Ring closure to form dihydro-orotate Step 4: Oxidation of dihydro-orotate to orotate Dihidroorotate dehydrogenase (pyrimidine) NB: In eukaryotes the enzyme is bound to the inner mitochondrial membrane (ET/OxPhos)!

24 Step 5: Acquisition of ribose phosphate moiety β (the most catalytically proficient enzyme) OMP orotidine monophosphate UMP uridine monophosphate Step 6: Decarboxylation of OMP The last 2 enzymes of the pathway are contained on a single multifunctional protein in the cytoplasm

25 Synthesis of UTP and CTP Synthesis of pyrimidine nucleotide triphosphates is similar to purine nucleotide triphosphates Two sequential reactions catalyzed by specific nucleoside monophosphate (cytidylate) kinase and nucleoside diphosphate kinase: UMP + ATP <---> UDP + ADP UDP + ATP <---> UTP + ADP Cytidine nucleotides are synthesized from UMP. Before the uridine base can be converted to cytidine the UMP must be phosphorylated to UTP. C4 CTP is formed by amination of UTP by CTP synthetase In bacteria, amino group from ammonia

26 Regulation of pyrimidine de novo synthesis Bacteria: Regulated at Reaction 2 (ATCase) - Allosteric activation by ATP - Inhibition by CTP (in E. coli) or UTP (in other bacteria). Animals: Controled by carbamoyl phosphate synthetase II - Inhibited by UDP and UTP - Activated by ATP and PRPP Mammals: second control at OMP decarboxylase (competitively inhibited by UMP and CMP) PRPP affects rate of OMP production (ADP & GDP inhibit PRPP synthesis) Bacteria have a salvage pathway for the pyrimidine bases. It is unclear whether humans have it.

27 Review of nucleotide biochemistry 1. FOLATE BIOCHEMISTRY 2. PYRIMIDINE SYNTHESIS 3. PURINE SYNTHESIS 4. CONVERSION OF RIBO- TO DEOXYRIBO-NUCLEOTIDES

28 Differences between RNA and DNK The four ribonucleotides obtained from the biosynthesis pathways (AMP, GMP, UMP, and CTP) are reduced to the deoxyribonucleotides needs for DNA synthesis. How?

29 Overview of dntp biosynthesis (not de novo synthesis from deoxyribose-containing precursors) Ribonucleotide reductases (RNRs)* reduce all four ribonucleotides to their deoxyribose derivatives. NB: A free radical mechanism in the ribonucleotide reductase reaction. Three classes of RNR enzymes in nature (different prosthetic group): Class I: Fe or Mn, Tyr radical, uses NDPs (aerobic prokaryotes and eukaryotes) Class II: adenosylcobalamin, uses NTPs (cyanobacteria, some bacteria, Euglena) Class III: SAM and Fe-S, Gly radical, uses NTPs (anaerobic prokaryotes) * Enzyme family responsible for removal of hydroxyl groups from nucleic acids

30 Structure of rndp reductase (E. coli, Class I) Enzyme is associated with small electron transport chain: Enzyme itself and small disulfide containing protein(s). (Heterotetramer) The most common form (class I) is an 2 2 dimer The two subunits form the large subunit of the protein (R1), which contain active site The two subunits make up the small subunit (R2), which contains the free radical Enzyme contains an activity site, a specificity site, and the catalytic site.

31 Reduction of a ribonucleoside diphosphate by rndp reductase NB: Electrons ultimately come from NADPH and are delivered to ribonucleotide reductase by either thioredoxin or glutaredoxin. Thioredoxin is a small protein that carries two reversibly oxidizable sulfhydryl (-SH) groups that participates in a wide variety of reduction/oxidation reactions. Glutaredoxins are small redox enzymes. In contrast to thioredoxins, which are reduced by thiredoxin reductase, glutaredoxins are reduced by the oxidation of glutathione.

32 Ribonucleotide reductase has a unique control mechanism (to assure that the deoxyribonucleotides are synthesized in adequate and balanced amounts) The activity site turns the enzyme ON or OFF : - When the activity site is occupied by ATP the enzyme is turned ON. - When the activity site is occupied by datp the enzyme is turned OFF. The specificity site* controls which nucleotide will be reduced: - if specificity site is occupied by ATP or datp then CDP or UDP is reduced. - if specificity site is occupied by dttp then GDP is reduced. - if specificity site is occupied by dgtp then ADP is reduced. * Complex feedback network (krajnje pojednostavljeno!!!) The catalytic site performs the reduction.

33 Proposed reaction mechanism for ribonucleotide reductase 1. Free radical (X ) abstracts H atom from C3 of the substrate 2. Acid-catalyzed cleavage of the C2 -OH bond 3. Radical mediates stabilization of the C2 cation (unshared electron pair) 4. Radical-cation intermediate is reduced by redox-active SH- pairdeoxynucleotide radical 5. 3 radical re-abstracts the H atom from the protein to restore the enzyme to the radical state. Reducing equivalents are supplied by the formation of an enzyme disulfide bond.

34 dntps made by phosphorylation of dndp Reaction is catalyzed by nucleoside diphosphate kinase (NDPK)* (same enzyme that phosphorylates NDPs): dndp + ATP dntp + ADP Can use any NTP or dntp as phosphoryl donor - Prokaryotic NDPK forms a functional homotetramer - There are two isoforms of NDPK in humans: A and B. Both have very similar structure, and can combine in any proportion to form functional NDK hexamers * catalyze the exchange of phosphate groups between different nucleoside diphosphates

35 Deoxyuridylate nucleotides are never incorporated into DNA Two mechanisms assure that the deoxyuridylate nucleotides are not incorporated into DNA: (a) First, the enzyme deoxyuridine triphosphate diphosphohydrolase (dutpase) rapidly converts any dutp that is formed to dump. dutp + H 2 O dump + PP i - minimized [dutp] prevents incorporation of uracil into DNA (b) Second, the dump is rapidly and quantitatively converted to dtmp (enzyme thymidylate synthase). - Thymine synthesis

36 Thymine synthesis The enzyme thymidylate synthase: (a) Catalyzes the transfer of the one carbon methylene fragment from TH 4 to C5 of uridine, and (b) It simultaneously reduces the one carbon fragment to a methyl group NB: N5,N10-methylene-THF as methyl donor Dihydrofolate is useless to the cell. It must be reduced to TH4, if cellular metabolism is to be maintained, by dihydrofolate reductase. NADPH and a H + ion donates the hydrogens and electrons necessary for the reaction. Once the TH4 is reformed it accepts a one carbon fragment from Ser or Gly and it is ready for the next cycle of reactions. NB: Regeneration of coenzyme The enzyme dihydrofolate reductase was the first target for cancer chemotherapeutic agents.

37 Catalytic mechanism of thymidylate synthase (highly conserved homodimeric protein) 1. Enzyme Cys thiolate group attacks C6 of dump (nucleophile). 2. C5 of the enolate ion attacks the CH 2 group of the imium cation of N 5,N 10 - methylene-thf. 3. Enzyme base abstracts the acidic proton at C5, forms methylene group and eliminates THF cofactor. 4. Migration of the N6-H atom of THF to the exocyclic methylene group to form a methyl group and displace the Cys thiolate intermediate.

38 2. Salvage pathway (for biosynthesis of purines) Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis can be directly converted to the corresponding nucleotides The significance of salvage pathway: - Save the fuel (the synthesis of purines is energy expensive) - Some tissues and organs (brain and bone marrow) are only capable of synthesizing nucleotides by salvage pathway Two phosphoribosyl transferases are involved: APRT (adenine phosphoribosyl transferase) for adenine. HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine. NB: Diverse in character and distribution!

39 Purine salvage pathway Absence of activity of HGPRT leads to Lesch-Nyhan syndrome (giht) Loss of HGPRT leads to elevated PRPP levels and stimulation of de novo purine synthesis

40 Salvage and de novo pathways to thymine nucleotides Bacteria have a salvage pathway for the pyrimidine bases. It is unclear whether humans have it.

41 Overview of nucleotides catabolism Excess purines and pyrimidines, originating from ingested nucleotides or from routine turnover of cellular nucleic acids, are catabolized. Most intracellular purine bases are salvaged and pyrimidine salvage probably occurs. Purine breakdown yields (except in part in muscle) only waste products that must be excreted (uric acid). Pyrimidines breakdown yields molecules (fatty acid intermediates) that can enter metabolism for energy generation

42 Simplified purine catabolism = uric acid (2,6,8-trioxypurine) The end product of purine metabolism

43 The major pathways of purine catabolism (animals) Adenosine and deoxyadenosine are not degraded by purine nucleoside phosphorylase (PNP) but are deaminated by adenosine deaminase (ADA) and AMP deaminase in mammals. R1P is isomerized by phosphoribomutase to R5P (precursor to PRPP) All pathways lead to formation of uric acid! Xanthine oxidase resides in lysosomes and peroxisomes In mammals, the uric acid is usually oxidized to allantoin. Intermediates could be intercepted into salvage pathways.

44 The purine nucleotide cycle generates fumarate The deamination of AMP to IMP, when combined with the synthesis of AMP from IMP, has the net effect of deamination aspartate to yield fumarate. This purine nucleotide cycle has an important metabolic role in skeletal muscle, in order to increase the concentration of Krebs cycle intermediates.

45 The proposed catalytic mechanism of adenosine deaminase (ADA) 1. Zn 2+ polarized H 2 O molecule nucleophilically attacks C6 of the adenosine. His is general base catalyst, Glu is general acid, and Asp orients water. 2. Tetrahedral intermediate decomposes by elimination of ammonia. 3. Product is inosine in enol form (assumes dominant keto form upon release from enzyme). Genetic defects in ADA kill lymphocytes (high levels of datp inhibits ribonucleotide reductase - no other dntps)

46 Simplified pirimidine catabolism The "end" product of pirimidine metabolism: β-amino acids

47 The major pathways of pyrimidines catabolism (animals) Pyrimidines degradation to their component bases! Happen (again!) through dephosphorylation, deamination, and glycosidic bond cleavage. Uracil and thymine broken down by reduction (vs. oxidation in purine catabolism) * Malonyl-CoA is a precursor of fatty acid synthesis, and methylmalonyl-coa is converted to the citric acid cycle intermediate succinyl-coa Energy metabolism!

48 Summary of nucleotide metabolism

49 Biosynthesis of NAD(P) + (Vitamine B3) Biosynthesis of CoA (Vitamine B5) Obnoviti/naučiti formule (ključnih) kofaktora/koenzima u metabolizmu!