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Amino acid catabolism; Nucleic acid chemistry Andy Howard Introductory Biochemistry 29 April 2008 What we’ll cover Amino acid catabolism Nucleosides Urea cycle Degradation products Nucleotides Interconversions Oligo- and Specifics polynucleotides Reactions Cellular localization Nucleic acid chemistry Pyrimidines: C, U, T Purines: A, G Amino Acid metabolism Duplex DNA Helicity RNA structure types p. 2 of 65 29 April 2008 What do we do with amino acids? Obviously a lot of them serve as building-blocks for protein and peptide synthesis via ribosomal mechanisms Also serve as metabolites, getting converted to other compounds or getting oxidized as fuel Most amino acid degradations begin with transaminations to make glutamate; the resulting alpha-keto acids are further metabolized Amino Acid metabolism p. 3 of 65 29 April 2008 Transaminations Generally two stages: amino acid + -ketoglutarate -keto acid + glutamate Glutamate + NAD+ + H2O -ketoglutarate + NADH + H+ + NH4+ Net reaction is amino acid + NAD+ + H2O -keto acid + NADH + H+ + NH4+ Amino Acid metabolism p. 4 of 65 29 April 2008 Glucogenic and ketogenic amino acids Degradation of many amino acids lead to TCA cycle intermediates or pyruvate Degradation of others leads to acetyl CoA and related compounds therefore these can be built back up to glucose; these are called glucogenic these cannot be built back up to glucose except via the glyoxalate shuttle these are called ketogenic Some amino acids are both! Amino Acid metabolism p. 5 of 65 29 April 2008 Glucogenic amino acids Amino acids that can be catabolized to produce building blocks that lead to glucose without help of glyoxalate pathway Most produce succinate, succinyl CoA, fumarate, a-ketoglutarate, or oxaloacetate Amino Acid metabolism p. 6 of 65 29 April 2008 Ketogenic amino acids These do not produce TCA cycle intermediates, but rather produce acetyl CoA or its close relatives Can be built back up into fats or ketone bodies Amino Acid metabolism p. 7 of 65 29 April 2008 Serine-based metabolites Serine is a building block for sphinganine and therefore for sphingolipids Serine also leads to phosphatidylserine, which is important by itself and can be metabolized to phosphatidylethanolamine and phosphatidylcholine Amino Acid metabolism p. 8 of 65 29 April 2008 Serine degradation Two paths for degrading serine: PLP-dependent serine dehydratase simply deaminates ser to pyruvate; this enzyme is like trp synthase More common: SHMT transfers hydroxymethyl group to THF, leaving glycine; we’ve seen that one as a biosynthetic enzyme for making glycine Amino Acid metabolism p. 9 of 65 Serine dehydratase PDB 1P5J 41 kDa monomer human 29 April 2008 Glycine-based metabolites porphobilinogen Glycine is a source for purines, glyoxalate, creatine phosphate, and (with the help of succinyl CoA) porphobilinogen, whence we get porphyrins, and from those we get chlorophyll, heme, and cobalamin Amino Acid metabolism p. 10 of 65 29 April 2008 Glycine cleavage system Glycine + H2O + NAD+ + THF NADH + H+ + HCO3- + NH4+ + 5-10-methyleneTHF Complex system: PLP, lipoamide, FAD prosthetic groups Lipoamide swinging arm works as in pyruvate dehydrogenase Amino Acid metabolism p. 11 of 65 T protein (aminomethyltransferase) of glycine cleavage system PDB 1V5V 88 kDa dimer Pyrococcus 29 April 2008 asp, glu, ala degradation Standard transmination converts aspartate to oxaloacetate with release of glutamate, which then can be deaminated to re-form ketoglutarate: asp + -kg oxaloacetate + glu glu + NAD+ + H2O -kg + NADH + H+ + NH4+ Deamination converts glutamate to ketoglutarate, as above Standard transamination converts alanine to pyruvate according to the same logic as asp Amino Acid metabolism p. 12 of 65 29 April 2008 All three of these are glucogenic! -ketoglutarate and oxaloacetate are TCA cycle intermediates Pyruvate feeds the TCA cycle in ways that can lead to glucose Amino Acid metabolism p. 13 of 65 29 April 2008 Degradation of asn, gln Asparagine and glutamine are deaminated to asp and glu Thus they lead to oxaloacetate and -ketoglutarate, respectively So they’re glucogenic The initial hydrolyses (deaminations) are catalyzed by asparaginase and glutaminase Amino Acid metabolism p. 14 of 65 Asparaginase PDB 1O7J 144 kDa tetramer Erwinia chrysanthemi 29 April 2008 Arginine degradation Arginine is hydrolyzed to urea and ornithine as part of the urea cycle; enzyme is arginase PLP-dependent enzyme converts ornithine to glu semialdehyde That’s oxidized to glutamate Amino Acid metabolism p. 15 of 65 Arginase PDB 2AEB 212 kDa hexamer Dimer shown Human 29 April 2008 Proline degradation Proline oxidized back to Pyrroline 5-carboxylate 1- O2 is oxidizing agent different enzyme from forward reaction Proline dehydrogenase PDB 2EKG 72 kDa dimer Thermus thermophilus Ring opened non-enzymatically to form glutamate semialdehyde; see arginine Amino Acid metabolism p. 16 of 65 29 April 2008 urocanate Histidine degradation 3 reactions from histidine to N-formiminoglutamate; first (HAL) makes urocanate from histidine Tetrahydrofolate-dependent reaction produces glutamate and 5formiminoTHF 5-formiminoTHF is enzymatically deaminated to 5,10-methyleneTHF, which can be used in purine synthesis, etc. Amino Acid metabolism p. 17 of 65 Histidine-ammonia lyase PDB 1GKM 224 kDa tetramer monomer shown Pseudomonas putida 29 April 2008 How are we doing so far? We did ser and gly first because they’re so important Then we’ve done a whole bunch that connect up to glutamate (or asp): asp, glu, ala, asn, gln, arg, pro, his So we’re halfway through. Amino Acid metabolism p. 18 of 65 29 April 2008 Threonine degradation Several pathways (fig. 17.29) Major one: oxidize threonine to 2-amino-3-ketobutyrate 2-amino-3-keto-butyrate reacts with HS-CoA to form acetyl CoA and glycine So this one is ketogenic Other pathways are glucogenic Amino Acid metabolism p. 19 of 65 Threonine dehydrogenase PDB 2DFV 115 kDa trimer Pyrococcus 29 April 2008 Valine degradation (fig. 17.30, center) Valine transaminated to -ketoisovalerate Branched-chain -keto acid dehydrogenase (TTP, Lipoamide): -ketoisoavalerate + NAD+ + HS-CoA a-ketoisovaleryl CoA Next reaction (acyl CoA dehydrogenase) 2-methyl-1-propenyl CoA + NADH + CO2 Product undergoes 4 reactions to propionyl CoA and thence to succinyl CoA: glucogenic Amino Acid metabolism p. 20 of 65 PDB 2VBF 125 kDa dimer Lactococcus 29 April 2008 Isoleucine and leucine degradation Same path but products are: Leucine’s products: acetyl CoA + acetoacetate: ketogenic Isoleucine: Acetyl CoA + propionyl CoA: ketogenic and glucogenic Amino Acid metabolism p. 21 of 65 29 April 2008 Methionine degradation A lot of methionine is turned into S-adenosylmethionine: Methyl donor Leaves behind S-Adenosylhomocysteine S-adenosylhomocysteine can be hydrolyzed to homocysteine and water Homocysteine can condense with serine to form cystathionine, which can yield cysteine and -ketobutyrate… and we know how to turn -ketobutyrate into propionyl CoA. So met is glucogenic. Amino Acid metabolism p. 22 of 65 29 April 2008 Cysteine degradation Cysteinesulfinate Most common: oxidation to cysteinesulfinate, which transaminates to form sulfinylpyruvate: cysteine + O2 cysteinesulfinate + H+ -sulfinylpyruvate undergoes nonenzymatic desulfuration to SO2 and pyruvate. So cysteine is glucogenic. Amino Acid metabolism p. 23 of 65 Cysteine dioxygenase PDB 2B5H 22 kDa monomer rat 29 April 2008 Tetrahydrobiopterin Phenylalanine Simple: phenylalanine gets hydroxylated to form tyrosine: phenylalanine + O2 tyrosine This is a tetrahydrobiopterindependent enzyme—a folate-like cofactor Amino Acid metabolism Phenylalanine hydroxylase PDB 1J8U 71 kDa dimer monomer shown human (residues 103-427) p. 24 of 65 29 April 2008 Phenylketonuria Usually associated with mutation in phenylalanine hydroxylase: Accumulated Phe phenylpyruvate Afflicts 1/15000 newborns Built-up phenylpyruvate causes irreversible mental retardation Type IV PKU related to deficiencies in enzymes that restore tetrahydrobiopterin (see fig. 17.33, bottom) Amino Acid metabolism p. 25 of 65 29 April 2008 homogentisate Tyrosine degradation Transaminated and mutated to homogentisate Three more reactions convert that to fumarate + acetoacetate So tyr (and phe) are both ketogenic and glucogenic Amino Acid metabolism Homogentisate dioxygenase PDB 1EYB 311 kDa hexamer Monomer shown Human p. 26 of 65 29 April 2008 Tryptophan degradation Tryptophan: need to open 2 rings! 8 reactions lead to alanine and ketoadipate; first is trp + O2 -> N-formyl-kynurenine Alanine gets transaminated to pyruvate -ketoadipate goes through 6 more reactions to acetyl CoA + 2CO2 So it’s ketogenic and glucogenic Amino Acid metabolism p. 27 of 65 Indoleamine 2,3dioxygenase PDB 2D0T 89 kDa dimer monomer shown human 29 April 2008 Lysine degradation (fig. 17.35) Condense lysine with -ketoglutarate to form saccharopine That’s deglutamated (?), oxidized, and transaminated to -ketoadipate Six reactions degrade that to 2 acetyl CoA molecules plus 2 CO2 Purely ketogenic Some bacteria decarboxylate it to cadaverine Amino Acid metabolism p. 28 of 65 Saccharopine dehydrogenase PDB 2AXQ 103 kDa dimer monomer shown Yeast 29 April 2008 The urea cycle: overview This is a significant pathway in the eukaryotic management of nitrogencontaining compounds It was also one of the first biochemical pathways to be carefully characterized—by Krebs and coworkers! Proceeds via ornithine & citrulline to urea and (in some organisms) uric acid Amino Acid metabolism p. 29 of 65 ornithine urea 29 April 2008 Making carbamoyl phosphate (fig. 17.37) Bicarbonate is phosphorylated to form Ammonia condenses with that to form carbamate and Pi Second ATP-phosphorylation forms carbamoyl phosphate Amino Acid metabolism p. 30 of 65 29 April 2008 Urea cycle itself In mitochondrion: carbamoyl phosphate condenses with ornithine to form citrulline Citrulline condenses with urea to form arginosuccinate Arginosuccinate is cleaved nonhydrolytically to fumarate and arginine Arginine yields urea and citrulline Citrulline re-enters cycle Amino Acid metabolism p. 31 of 65 29 April 2008 iClicker quiz: question 1 1. Glutamate + ammonia glutamine + H2O is only slightly endergonic (Go’ = +14 kJ mol-1), yet it is coupled to ATP hydrolysis. Why? (a) You can never run a reaction with a positive Go’ (b) [glutamate] ~ [glutamine] in the cell (c) If you heat the substrates, they disintegrate (d) ammonia is toxic in the absence of ATP Amino Acid metabolism p. 32 of 65 29 April 2008 iClicker quiz #2 2. Which ribosomal amino acid’s biosynthesis is closely associated with the urea cycle? (a) alanine (b) serine (c) ornithine (d) arginine (e) none of the above. Amino Acid metabolism p. 33 of 65 29 April 2008 6 5 Pyrimidines N 4 2 N 3 Single-ring nucleic acid bases pyrimidine 6-atom ring; always two nitrogens in the ring, meta to one another Based on pyrimidine, although pyrimidine itself is not a biologically important molecule Variations depend on oxygens and nitrogens attached to ring carbons Tautomerization possible Note line of symmetry in pyrimidine structure Amino Acid metabolism p. 34 of 65 1 29 April 2008 H N O Uracil and thymine Uracil is a simple dioxo derivative of pyrimidine: 2,4-dioxopyrimidine Thymine is 5-methyluracil Uracil is found in RNA; Thymine is found in DNA We can draw other tautomers where we move the protons to the oxygens Amino Acid metabolism p. 35 of 65 O HN uracil HN O N H thymine 29 April 2008 O H N O Tautomers Lactam and Lactim forms Getting these right was essential to Watson & Crick’s development of the DNA double helical model Amino Acid metabolism HN O NH O uracil - lactam H N O uracil - lactim HN HN O N H thymine - lactam O O N thymine - lactim p. 36 of 65 29 April 2008 OH H N O Cytosine NH2 N cytosine This is 2-oxo,4-aminopyrimidine It’s the other pyrimidine base found in DNA & RNA Spontaneous deamination (CU) Again, other tautomers can be drawn Amino Acid metabolism p. 37 of 65 29 April 2008 Cytosine: amino and imino forms Again, this tautomerization needs to be kept in mind H N O NH2 N cytosine -amino form Amino Acid metabolism H N O NH N cytosine -imino form p. 38 of 65 29 April 2008 7 6 5 1N N 3 8 4 2 Purines H N N 9 Derivatives of purine; again, the purine root molecule isn’t biologically important Six-membered ring looks a lot like pyrimidine Numbering works somewhat differently: note that the glycosidic bonds will be to N9, whereas it’s to N1 in pyrimidines Amino Acid metabolism p. 39 of 65 29 April 2008 Adenine This is 6-aminopurine Found in RNA and DNA We’ve seen how important adenosine and its derivatives are in metabolism Tautomerization happens here too NH NH2 H N N N N adenine - amino form Amino Acid metabolism H N HN N N adenine - imino form p. 40 of 65 29 April 2008 Guanine This is 2-amino-6-oxopurine Found in RNA, DNA Lactam, lactim forms OH O H N H N N HN H2N N guanine - lactam Amino Acid metabolism N H2N N N guanine - lactim p. 41 of 65 29 April 2008 O HO Nucleosides NR1R2 OH HO N-glycoside of ribofuranose As mentioned in ch. 8, these are glycosides of the nucleic acid bases Sugar is always ribose or deoxyribose Connected nitrogen is: N1 for pyrimidines (on 6-membered ring) N9 for purines (on 5-membered ring) Amino Acid metabolism p. 42 of 65 29 April 2008 Pyrimidine nucleosides Drawn here in amino and lactam forms OH OH HO HO OH O N H2N N OH O O N H cytidine Amino Acid metabolism O N O uridine p. 43 of 65 29 April 2008 Pyrimidine deoxynucleosides OH OH H H OH O N O N H OH O OH 2'-deoxyuridine O N H OH N O 2'-deoxythymidine O N H2N O N O deoxycytidine Amino Acid metabolism p. 44 of 65 29 April 2008 A tricky nomenclature issue Remember that thymidine and its phosphorylated derivatives ordinarily occur associated with deoxyribose, not ribose Therefore many people leave off the deoxy- prefix in names of thymidine and its derivatives: it’s usually assumed. Amino Acid metabolism p. 45 of 65 29 April 2008 Purine nucleosides Drawn in amino and lactam forms NH2 O N N N HN N N H2N N N O O HO HO OH OH HO HO guanosine adenosine Amino Acid metabolism p. 46 of 65 29 April 2008 Purine deoxynucleosides O NH2 N N HN N N H2N N N N O O OH OH HO HO deoxyguanosine deoxyadenosine Amino Acid metabolism p. 47 of 65 29 April 2008 Chirality in nucleic acids Bases themselves are achiral Four asymmetric centers in ribofuranose, counting the glycosidic bond. Three in deoxyribofuranose Glycosidic bond is one of those 4 or 3. Same for nucleotides: phosphates don’t add asymmetries Amino Acid metabolism p. 48 of 65 29 April 2008 NH2 Monophosphorylated nucleosides N N We have specialized names for the 5’-phospho derivatives of the nucleosides, i.e. the nucleoside monophosphates: They are nucleotides N N O HO OO P HO O adenylate Adenosine 5’-monophosphate = AMP = adenylate GMP = guanylate CMP = cytidylate UMP = radiate Amino Acid metabolism p. 49 of 65 O- 29 April 2008 Deoxynucleotides O Similar nomenclature dAMP = deoxyadenylate dGMP = deoxyguanylate dCMP = deoxycytidylate dTTP (= TTP) = deoxythymidylate = thymidylate Amino Acid metabolism N HN H2N N N O OO OP HO O deoxyguanylate p. 50 of 65 29 April 2008 Di and triphosphates Phosphoanhydride bonds link second and perhaps third phosphates to the 5’-OH on the ribose moiety O N O H2N O O O P P P O N O O- O O- OH O- Mg2+ OH HO cytidine triphosphate Amino Acid metabolism p. 51 of 65 29 April 2008 Oligomers and Polymers Monomers are nucleotides or deoxynucleotides Linkages are phosphodiester linkages between 3’ of one ribose and 5’ of the next ribose It’s logical to start from the 5’ end for synthetic reasons Amino Acid metabolism p. 52 of 65 29 April 2008 Typical DNA dinucleotide Various notations: this is pdApdCp Leave out the p’s if there’s a lot of them! -O OP O O O -O N O- N P O O O O N -O P O HN O NH2 O N O Amino Acid metabolism p. 53 of 65 N NH2 29 April 2008 DNA structure Many years of careful experimental work enabled fabrication of double-helical model of double-stranded DNA Explained [A]=[T], [C]=[G] Specific H-bonds stabilize double-helical structure: see fig. 19.12 Amino Acid metabolism p. 54 of 65 29 April 2008 What does double-stranded DNA really look like? Picture on previous slide emphasizes only the H-bond interactions Fig.19.12 is better: shows the tilt of the sugars Planes of the bases are almost perpendicular to the helical axes on both sides of the double helix Amino Acid metabolism p. 55 of 65 29 April 2008 Sizes (see fig. 19.14) Diameter of the double helix: 2.37nm Length along one full turn: 10.4 base pairs = pitch = 3.40nm Distance between stacked base pairs = rise = 0.33 nm Major groove is wider and shallower; minor groove is narrower and deeper Amino Acid metabolism p. 56 of 65 29 April 2008 What stabilizes this? Variety of stabilizing interactions Stacking of base pairs Hydrogen bonding between base pairs Hydrophobic effects (burying bases, which are less polar) Charge-charge interactions: phosphates with Mg2+ and cationic proteins Amino Acid metabolism p. 57 of 65 Courtesy dnareplication.info 29 April 2008 How close to instability is it? Pretty close. Heating DNA makes it melt: fig. 19.17 The more GC pairs, the harder it is to melt Weaker stacking interactions in A-T One more H-bond per GC than per AT Amino Acid metabolism p. 58 of 65 29 April 2008 iClicker quiz 3. What positions of a pair of aromatic rings leads to stabilizing interactions? (a) Parallel to one another (b) Perpendicular to one another (c) At a 45º angle to one another (d) Both (a) and (b) (e) All three: (a), (b), and ( c) Amino Acid metabolism p. 59 of 65 29 April 2008 Final iClicker question! 4. Which has the highest molecular mass among the compounds listed? (a) cytidylate (b) thymidylate (c) adenylate (d) adenosine triphosphate (e) they’re all the same MW Amino Acid metabolism p. 60 of 65 29 April 2008 Base composition for DNA As noted, [A]=[T], [C]=[G] because of base pairing [A]/[C] etc. not governed by base pairing Can vary considerably (table 19.2) E.coli : [A], [C] about equal Mycobacterium tuberculosis: [C] > 2*[A] Mammals: [C] < 0.74*[A] Amino Acid metabolism p. 61 of 65 29 April 2008 Supercoiling Refers to levels of organization of DNA beyond the immediate double-helix We describe circular DNA as relaxed if the closed double helix could lie flat It’s underwound or overwound if the ends are broken, twisted, and rejoined. Supercoils restore 10.4 bp/turn relation upon rejoining: see fig. 19.19. Amino Acid metabolism p. 62 of 65 29 April 2008 Supercoiling and flat DNA Diagram courtesy SIU Carbondale Amino Acid metabolism p. 63 of 65 29 April 2008 Ribonucleic acid We’re done with DNA for the moment. Let’s discuss RNA. RNA is generally, but not always, singlestranded The regions where localized base-pairing occurs (local double-stranded regions) often are of functional significance Amino Acid metabolism p. 64 of 65 29 April 2008 RNA physics & chemistry RNA molecules vary widely in size, from a few bases in length up to 10000s of bases There are several types of RNA found in cells Type %%turn- Size, RNA over by mRNA 3 25 50-104 tRNA 15 21 55-90 rRNA 80 50 102-104 sRNA 2 4 30-103 Amino Acid metabolism Partly DS? no yes no ? Role protein template aa activation transl. catalysis & scaffolding various p. 65 of 65 29 April 2008