Nucleotides rata _and Nucleic Acids Essential Question Nucleotides and’ nucleic acids are compounds containing nitrogen bases (aromatic cel structures possessing nitrogen atoms) as part of thir structure. Nucleotides are essential to cellular metabolism, and nucleic acids are the molecules of genetic infor: imation storage and expression. What are the structures of the nucleotides? Hove are nucleotides joined together to form nucleic acids? How is information stored jn nucleic acids? What are the biological functions of nucleotides and nucleic acids? Nucleotides and nucleic acids are biological molecules that possess hetero- cyclic nitrogenous bases as principal components of their structure. The bio- © chemical roles of nucleotides are mumerous; they participate as essential in- france Chek (/gh and Janes Watson fet pork termediates in virtually all aspects of cellular metabolism. Serving an even out ealures of the rede for the structure of DNA, more central biological purpose are the nucleic acids, the elements of hered- _ ity and the agents of genetic information transfer. Just as proteins are linear polymers of amino acids, nucleic acids are linear polymers of nucleotides. We have discovered the seoret of lie! Like the leteers in this sentence, the orderly sequence of nucleotide residues _ Proclamation by Hanes Il, C. Crick to patrons ina mucleic acid can encode information. The two basic kinds of nucleic acids __ the Eagle, pub in Cambridge, Englanc | are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Complete _“") __Iyelrolsis of nucleic acids liberates nitrogenous bases, a fivecarbon sugar, Fe ind phosphoric acid in equal amounts, The five-carbon sugar in DNA is 2 _ deoxyribose; in RNA, ivis ribose. (See Chapter 7 for a detailed discussion of Key Questions © sugars and other carbohydrates.) DNA is the repository of genetic informa- 10-4 Whats ihe Stace and Chemisty ‘of Nivogenous bases? 10.2. What Are Nucesides? 10.3. Whats the Stuctre and chemistry ‘of Hudoties? Ree an charts BA sein the eapteaton of information throogh the procee of eanacription snd ansaton (Pgore 101) A terentng exception to this ale iat some irae have thet geneti nfo: maton sored RNA : sna whaerenc Aa? ‘This chapter describes the chemistry of nucleotides and the major classes of | _ hues acids Chaper 11 present methods for determination ofmactee aig 108. Wate ie Difleen Cheol abc ‘Sinacycracrare futile ud aequancin) and desedbesthe higher adeno sy sete toydont nucleic acid structure. Chapter 12 introduces the molecular biology of recombinant 6 ee Nad Acs Stomp to thobss DNAS the sonsution and uct of novel DNA molest aatentled by com. ining segments from other DNA molecules, What Is the Structure and Chemistry of Nitrogenous Bases? [= ing two nitrogen atoms (Figure 10.22). The atoms are numbered in a clockwise _fishion, as shown in Figure 10.2. The purine ring system consists of two rings ‘of atoms: one resembling the pyrimidine ring and another resembling the _ imidazole ring (Figure 10.2b). ‘The nine atoms in this fused ving system are | humbered according to the convention shown. |. The pyrimidine sing sytem is planar, whereas the purine system deviates _ Somewhat from planarity in having a slight pucker between is imidazole and ‘Pyrimidine portions, Both are relatively insoluble in water, a8 might be ex- ected from their pronounced aromatic character. BiochemistryZ)Now™ Test yoursef on ‘these Key Questions a BochemistyNow at http:lchemistry brookscole.com/ggb3FIGURE 10.1 ‘The fundamental proces of informa tion transfer ince. (2) Tformation encoded in the nucleotide sequence of DNA ie transcribed through ‘Suthesis of RNA molecule whose sequence is reat by the DNA sequence. (2) As the sxquence bf this RNA fread (as groups of three conseeative nucleotides) bythe protein sythesi machinery, js wanslated into the sequence of amino acids in [protein This information transfer system i encap- [Entec in the dogins. DNA -> RNA protein. Biochemistry Now" Goto BiacheristryNow and cick ichetyintratve to lear he stu tures ofthe common pines an pines. », ra ‘The pyrimidine ring "The purine ring estem FIGURE 10.2 (2) The primidine rng ystems: by ‘convention, aoms are numbered as indlated {@) The purine ring stem, atoms numbered 26 shown, Replication, DNA replication yields wo DNA molecules Wdentcal to the ‘original one, ensuring transmission Sf genetic information to daughter cele with exceptional dey, ‘Transelption “The sequence of bases in DNA is recorded a sequence of ‘complementary bases ina sgle- Stranded mRNA molecule, ‘Teansation ‘Threebase codons on the mRNA comesponding to specific amino aids Givect the sequence of building 2 protein, ‘These codons ate recognized bby ekNAs (transfor RNAS) carrying the appropriate amino acids. Ribosomes ‘Auached are the *machinery for protein faming acid syachenis it Growing peptide chaln Prot Three Pyrimidines and Two Purines Are Commonly Found in Cells ‘The common naturally occurving pyrimidines are cytosine, uracil, and thymine (Gmethyluracil) (Figure 10.8). Cytosine and thymine are the pyrimidines pi cally found in DNA, whereas eytosine and uracil are common in RNA. To view this generality another way, the uracil component of DNA occurs as the 5-mnethy! variety thymine. Various pyrimidine derivatives, such as dibydrouracil, are pres: ent as minor constituents in certain RNA. molecules. "Adenine (Gamino purine) and guanine (2-amnino-6-oxy purine), the two com- ‘mon purines, are found in both DNA and RNA (Figure 10.4). Other naturally occurring purine derivatives include hypoxanthine, xanthine, and urie acid (Fi ure 10.5), Hypoxanthine and xanthine are found only rarely as constituents of auclei¢ acids. Uric acid, the most oxidized state for a purine derivative, is never found in nucleic acids H a a Gytosine rae ‘Thine @oxy-tamine (oxy-tony Qoxy-soxy Pyridine) pytimicine) Smell pyrimidine) FIGURE 10.3 The common pyrimidine batercytosine, ural, and dhymine—in the tantomerie forms predominant at p37.40.1 Wats the Structure and Chemisty of Kogencus Bases Adenine ‘amino purine) Gaminooxy purine) FIGURE 10.4 ‘The common purine basesadenine and guanine—in dhe tautomeric forms pre- dominant 2¢ pT The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature ‘The aromaticity of the pyrimidine and purine ring systems and the electron- rich nature of their OH and —NHf substituents endow them with the ca- pacity to undergo Keto-enol tautomeric shifts. ‘That is, pyrimidines and purines exist as tautomeric pairs, as shown in Figure 10.6 for uracil. The keto tautomer is called a Inetam, whereas the enol form is a lactim, The lactam form vastly predominates at neutral pH. In other words, pX, values for ring ni- trogen atoms I and 3 in uracil are greater than 8 (the pX, value for N3 is 9.5) (Table 10.1). In contrast, as might be expected from the form of cytosine that, predominates at pH 7, the pX, value for N-8 in this pyrimidine is 4.5. Similar, kKeto-enol tautomeric forms can be represented for purines, as given for gua- nine in Figure 10.72 Here, the pK, value is 9.4 for Nel and less than 8 for N3. These pX, values specify whether hydrogen atoms are associated with the various ring nitrogens at neutral pH. As such, they are important in det mining whether these nitrogens serve as H-bond donors or acceptors. Hydro~ gen bonding between purine and pyrimidine bases js fundamental to the bio- logical functions of nucleic acids, as in the formation of the double-helix structure of DNA (see Section 10.5), The important functional groups parti ting in PE-bond formation are the amino groups of cytosine, adenine, and guanine; the ring mitrogens at position 3 of pyrimidines and position 1 of purines; and the strongly electronegative oxygen atoms attached at position 4of uracil and thymine, position 2 of cytosine, and position 6 af guanine (see Figure 10.20) ‘Another property of pyrimidines and purines is their strong absorbance of ultraviolet (UV) light, which is also a consequence of the aromaticity of their heterocyclic ving structures. Figure 10.8 shows characteristic absorption spec- tia of several of the common bases of ncteic acids—adenine, uracil, cytosine, and guanine—in their nucleotide forms: AMP, UMP, CMR, and GMP (see Sec- tion 10,3). This property is particularly useful in quantitative and qualitative analysis of nucleotides and nucleic acids {he 2 4 and Bprimidine and purine amino groups cn undergo tutomeriam at wel, chang ing Kom amino to mina functions pe Based ok, Phosphate PK; Phosphate s-AMP 3.8 (NA) 08 61 soup 9A (NAL) 07 61 24 (N-) cmp 45(N3) 08 63 scomp 95 (N38) 10 64 an Use acid FIGURE 10.5 Oxner naturally oceurting pusine erivatines-“hypoxanthine, xanthine, and ure acid, : Wa poe h a FIGURE 10.6 ‘The keto-enol automeriaton af ‘rac FIGURE 10.7 The tantomerization of the purine sutnin.312 Chapter 10 Nudeotites and Nuc Adis aN von 10 4 = ant? es os fos fos es an oa yey pate ane os oa 0 Pemneest eam Sen so Phamnmeneen ONO ROB 3 war me0 sm) ao aloraeo a0" ° G0" SOT a Sa vise am ° arden vee vowing FIGURE 10.8 The UY absorption spect ofthe ‘common ribonaeletides. 10.2 | What Are Nucleosides? ea Nucleosides are compounds formed when a base is inked o a sugar The sagas of nucleosides are pentoses(five-carbon sugars, ee Chapter 7). Ribonudleosides contain the pentose D-ribose, whereas 2-deoxy ribose is found in deoxyeibom. cleosides. In both instances, the pentose isin the five membered ring form known 4s furanose: p-ibofuranose for ribontcieosides and 2leoxy-Dribofuranose for deoxyribonucleosdes (Figure 109). In nucleosides, these ribofuranose atoms are sso otto distinguish them from the ring atoms of the nitzogenous bases. (As we shall se, the seemingly minor diference of a hydroxyl group a the 2-postion has furreaching ffecs on the secondary structures aval able to RNA and DNA, aswell a their relative susceptibilities to chemical and en- aymatic hydrolysis) Tn nucleosides, the base is linked to the sugar via a glycoside bond (Fig ure 10.10). Glycosidic bonds by definition involve the earbony! carbon atom of the sugar, which in eylic structures is joined to the ring O atom. As discussed in Chapter 7, such carbon atoms are called anomeric. In nucleosides, the bond isan ‘Ngycoside because it connects the anomeric Cl’ to N-l of a pyrimidine or to N-9 ofa purine. Recall that glycosidic bonds can be either «or f, depending on their orientation relative to the anomeric Catom. Glycosdic bonds in nucleosides (and mucleatides, see following discussion) are always of the Peconfiguration, as represented in Figure 10,10. Nucleosides are named by adding the ending -idine to the root name of a pyrimidine of -esineto the root name ofa purine. The com mon nucleosides ate thus gtidine, uridine, thymidine, adenosine, and guanosine. Structures of the common ribonucleosides are shown in Figure 10.11. The nucle «side formed by hypoxanthine and ribose is inosine. Nucleosides Usually Adopt an Anti Conformation About ‘the Glycosidic Bond In nucleosides, rotation of the base about the glycosidic bond is sterically bin- dered, principally by the hydrogen atom on the 2" carbon of the furanose- (This hindrance is most easily seen and appreciated by manipulating accurate me eo eet on Hob Con ont Poe H-C—On ‘Re OH ‘RH i - Nir weno Bie f on unas Fie rm of Hepa Skee, DH Sates FGURE 109 renresmcanessowrand viene == Rn Ranne” Bone BA Dennrefrte | aeoxyribae,ae rho . ’ Ka he ke De i " ( A Te Of Ss pine phone yond pyrimidine ond in pure oa ribonucteosides ‘bonueteosides FIGURE 10.10 .-Cycosiaic bonds fink nitrogenous bases and sugars to forma nucleosides molecular models of these structures.) Consequently, nucleosides (and nu- cleotides, see next section) exist in either of two conformations, designated syn and anti (Figure 10.12). For pyrimidines in the syn conformation, the oxygen substituent at position C-2 would lie in a sterically hindered position immedi- ately above the furanase ring; in the anti conformation, this steric interference is avoided, Consequently, pyrimidine nucleosides adopt the anti conformation. Purine nucleosides can assume either the syn or anti conformation, although the anti conformation is favored. In either conformation, the roughly planar furanose and base rings are not coplanar but lie at approximately right angles to one another. Nucleosides Are More Water Soluble Than Free Bases Nucleosides are much more water soluble than the free bases because of the hydrophilicity of the sugar moiety. Like glycosides (see Chapter 7), nucleosides are relatively stable in alkali. Pyrimidine nucleosides are also resistant to acid hydrolysis, but purine nucleosides are easily hydrolyzed in acid to yield the free ‘base and pentose, Nth ° ‘0 ° Hoct, 4 HOCH, HOH, Ka Ho aon ® (i " iH a (3 on on on on on OH Oysaine Uridine Adenosine (an On On ter Guasosine Taosin, an uncon FIGURE 10.11 The common ribonucleesides— cytidine, uridine, adenosine, and guanosine. Alo, inesne dram in ani conformation.soul 314 Chaptor 10 Nucleotides and Nucleic Ais ° ut © HOCK 9, HH FIGURE 10.12 Ros 1 the ey a i 1:12. Rotation around the please ‘ond is sterically hindered sn versus ai cone on on ou on 90! Guanesine 10.3 ‘A nucleotide results when phosphoric acid is esterified to a sugar —OH group of nucleoside. The nucleoside ribose ring fas three —OH groups available for esterification, at C2", C3!, and G5" (although 2 deoxyribose has only two). The vast majority of monomeric nucleotides in the cell are ribonucteotides having 5'-phosphate groups. Figure'l0.13 shows the structures of the common four rbo- nucleotides, whose formal names arc adenosine 5" monophosphate, guanosine ‘Stanonophosphate, cytidine 5'-monophosphate, and uridine 5'-monophosphate, These compounds are more often referred to by their abbreviations: 5"-AMP, 5!-GMP, 5'-CMP, and 5’-UMP, or even more simply as AME, GMP, CMB, and UMP. Nucleoside 8’phosphates and nucleoside 2" phosphates (8'NMP and .Y-NMP, where N isa generic designation for “nucleoside”) are uncommon, cept as products of nucleic acid hydrolysis. Because the pK, value for the first dis aration in ntcleorides are shown. sociation of a proton from the phosphoric acid moiety is 1.0 or less (Table 10.1), the nucleotides have acidic properties. This acidity is implicit in the other names by which these substances are known—adenylic acid, guanylic acid, cytidylic acid, Adenosine: A Nucleoside with Physiological Activity rate, Adenosine is licensed and marketed as Adenocard to treat supraventricular tachycardia In addition, adenosine is implicated in sleep regulation. Dur ing periods of extended wakefulness, extracellular adenosine For the most part, nucleosides have no biological role other than to serve az component parts of nucleotides. Adenosine isa rare exception. In mammals, adenosine functions as an autocoid, or “local hormone," and as a neuromodulator. This nucleoside cir- alates in the bloodstream, acing Hocaly on specie cel to Ituence such diverse piysolgiesl ppenemens a blood seve dilaton, smooch muscle contraction, neuronal dicharge, new transmitter release, and melabolian of fat. For example, when Tousces work hard they felese adenosine, caring the sr rounding blood wesc edit, which tur increases the flow tf blood and its delvery of Oy and nutrients to the sce. a different autocoid vole, adenosine acts in regulating heartbeat ‘The natal shythm of the hear controll hy a pacemaker thesinoat il nade, whlch cyclical sends a wave electra ex tiation to the heat muscles. By blocking the flaw of electrical Current adenosine slows the hear sat. Supraveteta lahyar Sea heart condition characterized by rapid hearer Jeclon of alenotine eas s'momentary interruption OF the rapid ele of contacion and rewores a normal heart ron tone T 187, ane: mbsf Spit eo po lege ins seme 27005-1268 oo Vege MK, 2002 Posie hed en ca ee 4730-78, Tevels rise as a result of metabolic activity in the brain, and this jerease promotes sleepiness, During sleep, adenosine levels, fall. Caffeine promotes wakefulness by blocking the interaction of extracellular adenosine with its neuronal receptors.* 9 Cis me, oe 7) Y CH caffeineal . 103 What ithe Stuctue ad Chemisty of Nuceosides? 315) A phosphoester bond ° Hees on Ftat oO HOH oO H 2 i @ a on on or on Adenosine nonophospat Guanosine8'monoghaspate er it or elena) (or CM or gua acid) Ny ° a 9 ° i : Hee = On 08t On F-ORH is 0 H " t Sit on on on ptne5' monophosphate ‘Uridine 5'monophowphate (er hator guage id) (or UND aruda) and uridylic acid. The pK, value forthe second dissociation, pK, is about 60, s0 at neutral pH or above, the net charge on a nucleoside monophosphate is ~2. Nucleic acids, which are polymers of nucleoside monophosphates, derive their ‘ame from the acidity ofthese phosphate groups. Cyclic Nucleotides Are Cyclic Phosphodiesters Nucleoside monophosphates in which the phosphoric acid is esterified to teo of the available ribose hydroxyl groups (Figure 10.14) are found in all cells. Forming oxo such ester linkages with one phosphate results in a cyclic phos- phodiester structure. 8,5"-eyclic AMP, often abbreviated cAMP, and its guanine ‘analog 3',8'-cyclic GMP, or GMP, are important regulators of cellular metab- clisin (see Parts $ and 4). Nucleoside Diphosphates and Triphosphates Are Nucleotides with Two or Three Phosphate Groups Additional phosphate groups can be linked to the phosphoryl group of a mu cleotide through the formation of phosphorie anhydride linkages, as shown in Figure 10.15. Addition of a second phosphate to AMP creates adenosine 5'- diphosphate, or ADP, and adding a third yields adenosine 5'-triphosphate, or ATP, The respective phosphate groups are designated by the Greek letters ¢, B, and 7, starting with the @phosphate as the one linked directly to the pentose. ‘The abbreviations GIB, GIP, and UTP represent the other corresponding nu- ‘leoside 5-triphosphates. Like the nucleoside 5'-monophosphates, the nucleo side 5'-diphosphates and 5’-triphosphates all occur in the free state in the cel, 4 do their deoxyribonucleoside phosphate counterparis, represented as dAMP, dADP, and dATP; GGMP, GDP, and dGTP; dCMP, ACDP, and dCTP; UME, dUDP, and dUTP; and dTMP, dTDP, and dTTP. NDPs and NTPs Are Polyprotic Acids ‘Nucleoside 5'-diphosphates (NDPs) and nucleoside 5'-triphosphates (NTPs) axe relatively strong pobyprotic acids in that they dissociate three and four pro- tons, respectively, from their phosphoric acid groups. The resulting phosphate Anueleoside "monophosphate SAME FIGURE 10.13 Structures ofthe four common "Hhonucleotides—AME, GMP, CME, ad UME— together with thee two sts of full names, For eam ple, adenosine B-monophesphate and adenyie ald ‘so showm is the nclecsde 8"AMP. 348:Cyuie GMP FIGURE 10.14 Stcuctuces ofthe eylic nucleotides ‘AMP and cGMP,316 Chapter 10 Nucleotides and Nuc Ads a. oh me fon Ca) W on OH Phosphate (%) + AMP (adenosine S'monophosphate) Water + ADP (adenosine S specific site. The pattern of inhibition by these nucleotides is competitive, thus ensuring that residual enzyme activity is expressed until sufficient amounts of both adenine and guanine nucleotides are synthesized. Glutamine phosphor bbosyl pyrophosphate amidotransferase is also sensitive to inhibition by the ghu- ‘amine analog azaserine (Figure 26.4). Azaserine has been used as an antite mor agent because it causes inactivation of glutamine-dependent enzymes in the purine biosynthetic pathway. Step 3 is carried out by glycinamide ribonucleotide synthetase (GAR synthetase) via its ATP-clependent condensation ofthe glycine carboxyl group with the amine of 5-phosphoriosy-B-anine (Figure 26.3). The reaction proceeds in two stages. First, the glycine carboxy! geoup is activated via ATP-dependent phosphorylation. Next, an amide bond is formed between the activated carboxyl group of glycine and the B-amine. Glycine contributes C-4, C-, and N-7 of the purine. Step 4 is the first of to THF-dependent reactions in the purine pathway. GAR transformylase transfers the "formyl group of N*°formyTHF to the free amino group of GAR to yield a-Nformylelyinamide ribonucleotide (PCAR)- ‘Thus, C-8 of the purine is “Formyliy” introduced. Although all of the atoms of the imidazole portion of the purine ring are now present, the ring is not closed until Reaction 6, Step 5 is catalyzed by FGAR amidotransferase (also know as FGAM syntheias). AfP-dependent transfer of the glutamine amido group to the C-4-carbonyl of FGAR yields formplgycinamidine ribonucleotide (RCAM), As a glutamine-dependent A Ni eormph-THF jtamine (amide)LET NTR f I b i ‘An elaborate ensemble of enaymatic reactions serves to intro- duce one-carbon units into THE and to interconvert the various oxidation states (see accompanying figure and Table 17.8). N= methyltetrahydrofolate can be oxidized directly to NS" methylenetetrahydrofolate, which can be further oxidized to AN" -methenyhetrahydrofolate, The N*formimino, N° formyl, and N° formyleetrahydrofolates can be formed from NN" methenyltetrahydrofolate (all of these being at the same oxidation level), or they ean be formed by one-carbon ad dition reactions from tetrahydrofolate itself. The principal Carbon wit ‘oxidation evel “romogptcine ee 12,0!" methylene THE Formate Tetrahydrofolate (THF) and One-Carbon Units Methionine mS med! THE ae injgine Oring PL wean) Forining Ga Formate & Foray ? g [saves aye ‘S F] +8 ( Nabe is i q NN a ie GBD vp oH” ape + ® a [ ow am A + ape + ® oa ( BAO He | reel Vaal ae a ay CH. x b be ese a ae naprt < ce oe Sy ace k NSsominioo THF gop N° formytTHE el NADPH. a ape +®. a 1" foray THE + s Ze oe a | me & ea 4 fa) IN. Lar Nope bent ® 1N°V! methenyl TH 262 How Do Cels synthesize Purines? 855 pathway for incorporation of one-carbon nits into tetrahydro- folate is the serine hydeoxymethyltransferase reaction (see Fig: ture 25.2), which converts serine to glyeine and forms N,N" methylenetetrahydrofolate. ‘The biosynthetic pathways for methionine, purines (Fig- ture 26.3), and the pyrimidine thymine (see Figure 26.27) all rely on the incorporation of one-carbon units from tetrahydro- folate derivatives. IV"¥formykTHE is the source of catbons 2 ‘and 8 of the purine ring and N?,W!&methylene-THE, the CH, group of the pyrimidine thymine. cS 1c a | Hah \ wo at 4H HN. H A The reactions that introduce one-carbon units into tevahydrofolate (THE) link seven different folate intersvediates that carry oneearbon unit in three different oxidation states (2, 0, ad 142). (Api for roy, Tay ect, 1986 Handbook of Vinny, No Wk Mara Den856 Chapter 26 The Symhesis and Degradation of Necleotides nosing mor Doragh THE ° i ON oN Noor aa iis Sa fy Ha ‘SAminomidarolecarhoxamide ribonucleotide (AICAR) coo 9 t t ra Ge Ay cog BN ual ‘Neuecinylo-Samincinidazole--carbossmide ‘ibomiceotide(SHICAR) o Ape + @ J SAICAR symetase Aspartate 4 ATP ™ 2006. y anes @ @ ay" Kew) AW: ae - oy Ano Carhoxyamtnoimidazolebonueootide (CAIR) on HO OH eon RihoseS-phosphate ace amspivease @ neh AM awet@ jaa ‘Aminolmidazole ibonuclotide (AIR) e Ch, H ivosesphospiata rropiemiotine KEG H o-P-0-P=0- Are AMP oneal i oo ‘Phosphoribosyha-pyrophosphate (PREP) Gruamine + AB, see anid & corner mise. Giusmate + Bay o_ my Ho HD an 0 On Pherphorbon amine ciycine + AP Girt GaReyninerse — @ appt @ ae (fie Nie eae Beto ‘wa Gm peace aay Pern 8 GAR tansformylase Q) mu : Ny sigan wee va ow ee eciea ae ate + Guanine + fiby BEN vemos @ ADP + Glutamate + @& H x ae" Fornylgycinamidine ibonuelotde (GANlachamistry@Now™ ACTIVE FIGURE 26.3 The dc novo pathway for pasine shes ‘The ist purine product ofthis pay, IMP (inosn‘ acid or inosine monophosphate), serves 484 precaraor to AMP and GMP Step 1 PRPP syuiesis fom ribose phosphate and ATP by “bose phoiphate prophesphokinse. Step 2 bPhosohorbonyB.--amin sess om {PPRPR, glace, snl HO by glutamine Phorporiboy pycophosphaseseadowansteae Step 3: Gyeinamide sboneeoude (GAR) symeds rom gicine, ATL, and Sphosphoribon famine by ecinamidesitomorleoide sntase Step 4 Formyihcnamide chorleoite Syess fom N'Wformy-THF and GAR by GAR transformglase. Step 5: Formylyeinamidine ‘Nbonucleotde (FGAMD syne from FGAR, ATR, glitanine, snd HO by FOAM symihetase (GAR amdoanserate). The other products are ADP, and gluamate Stop 6 Amini {azole sbonseleose (Al) apntesais achieved va the ATP-dependent clone ofthe nd: tole sng, a catalyzed by GAM cylae (AIR syndetase). (Note thatthe ig close changes the numbering stem.) Step 7: Caroxpurinolmidarole ibonncleasde (CAIR) synthesis or 0, ATR, and AIR by ATR crboxgate, Step NecelnploS-amincimidasole-t-carboramide ribonucleotide (SAICAR) synthesis rom aspartate, CATR, and ATP by SATCAR ayuda Stop 8 &-Aminoimdaote cabosamide bensletie (AICAR) frtaton bythe non {rej removal a marate from SAICAR. The enzyme is ademlomernar. Step 103 Foray bimiavole carboxamide rtontelotde (FAICAR) formation from ATCAR and Nome THE by AICAR wansformylase. Stop Il: Dehyraton/ring closure yeds the authenie pune ‘Shonucleotde IMP, The enayme STM sytase Test yourself onthe concepts inthis igure at thup/ chemist brootacolacom/agb3 enzyme, FGAR amidotwansferase is like glutamine phosphoribosyl pyrophosphate amidotransferase (Reaction 2), irreversibly inactivated by azaserine. The imino-N becomes N'3 of the purine, Step 6 is an ATP-dependent dehydration that leads to formation of the imi- dazole ring. ATP is used to phosphorylate the oxygen atom of the formyl group, activating it for the ring closure step that follows. Because the product is S-antinoimidazole ribonucleotide, or AIR, this enzyme is called AIR synthetase. In avian liver, the enzymatic activities for steps 8, 4, and 6 (GAR synthetase, CAR transformylase, and AIR synthetase) reside on a single, 110-kD multifunctional polypeptide. In step 7, carbon dioxide is added at the C-4 position of the imidazole ring by AIR carboxylase in an ATP-dependent reaction; the carbon of CO; will become C6 of the purine ring. The product is carbxyaminoimidazoleribonucieatide (CATR). In step 8, the aminoN of aspartate provides Nl through linkage to the C46 carboxyl function of GAIR. ATP hydrolysis drives the condensation of Asp With GAIR, The productis Nsuecinylo-S-aminaimidazole-4-carbosantde ribonuclentide (SAIGAR). SAICAR synthetase catalyzes the reaction. The enzymatic activities for + steps 7 and 8 reside on a single, bifunctional polypeptide in avian Tver Step 9 removes the four carbons of Asp as fumarate in a nonhydrolytic cleave age. The product is S-aminaimidazole-4-carboxamide ribonucleotide (STCAR); the ‘enzyme is adenylosuecinase (adenyosuecinate hase). Adenylosuccinase acts again jn that part of the putine pathwtay leading from IMP to AMP and takes its name from this latter reaction (sce following). AICAR is also an intermediate in the histidine biosynthetic pathway (sce Chapter 25), but because ATP is the pre~ ‘cursor to AICAR in that pathway, no net purine synthesis is achieved. Step 10 adds the formyl carbon of NMformyTHF as the ninth and last atom. necessary for forming the purine nucleus. The enzyme is called AICAR trans- Formylase; the products are THE and Nformylaminoimidazole-4-carboxamide ribon- deatde (FAICAP). Step 11 involves dehydration and ring closure and completes the initial phase of purine biosynthesis. The enayme is IMP eyclohydrolase (also known as IMP synthase and inosinicase). Unlike step 6, this ring closure does not require ATR. In avian lives, the enzymatic activities catalyzing steps 10 and 11 (AICAR ‘wansformylase and inosinicase) activities reside on 67-KD bifunctional polypep- tides organized into 1354D dimers, Note that 6 ATPs are required in the purine biosynthetic pathway from ribose- S,phosphate to IMP: one each at steps 1, 3,5, 6, 7, and 8, However, 7 high-energy phosphate bonds (equal to 7 ATP equivalents) are consumed because c-PREP formation in Reaction 1 followed by PP, release in Reaction 2 represents the loss of 2 ATP equivaients. 26.2. How Oo Cells Symes Puines? Araserine ‘Giatamine ° c-o-ay, i ° By 6 —cal,— cr, ost un, 387 ° at syns ° a \, FIGURE 26.4 ‘The structure ofanserine. Azseerine cts as an irreversible inhibitor of glutamine ‘dependent enaymes by covalently aaching to rueleophille groups inthe glutamine binding ste.