生物化學(xué)本科課件英文版講授chapter

1、CHAPTER 29 BIOSYNTHESIS OF NUCLEOTIDES Nucleotides play key roles in the following biochemical processes: As activated precursors of DNA and RNA核酸前體Their derivatives are activated intermediates in many biosyntheses, e.g. UDP-Glc and CDP-DAG and S-A Met 活性中間物ATP is a universal currency of energy in b

2、iological systems, GTP powers many movements of macromolecules能量通用形式Adenine nucleotides are components of 3 major coenzymes: NAD+, FAD, and CoA輔酶As metabolic regulators, e.g. cAMP and ATP 代謝調(diào)節(jié)物 Nucleotides are synthesized from simple building blocks (de novo synthesis從頭合成) or by the recycling of pre

3、formed bases (salvage synthesis節(jié)約利用途經(jīng)) Purines (Pur.) and pyrimidines (Pyr.) are built de novo from AA, FH4 derivatives, NH4+ and CO2 The PR moiety of ribonucleotides comes from PRPP, an activated donor Deoxyribonucleotides are synthesized by reduction of ribonucleotides(dNDP from NDP) Finally, dTMP

4、 is formed by methylation甲基化 of dUMP (UMP dUMP dTMP) Nucleotide analogs are valuable drugs in the treatment of cancers(P753), viral infections, autoimmune diseases, and genetic disorders such as gout痛風(fēng)癥(P757) The purine ring is synthesized from AA, FH4 derivatives, and CO2 The Pur Ring is assembled

5、de novo from several simple precursors 1. Gly (C-4, C-5, N-7) 2. Asp (N-1) 3. Gln (N-3, N-9)兩次 FH4 (C-2, C-8)兩次 CO2 (C-6) PRPP is the donor of the PR unit of nucleotides (Nt) PRPP provides PR portion for the synthesis of Pur and Pyr Nt, just as for that of Trp PRPP is synthesized from ATP and R-5-P,

6、 which is primarily formed by PPP PRPP synthetase catalyzes the transfer of the - pyrophosphoryl group of ATP to C-1 of R-5-P PRPP has an -configuration at C-1, the activated carbon atom -構(gòu)型The Pur ring is assembled on PR The committed step in the de novo synthesis of Pur Nt is the formation of 5-ph

7、osphoribosylamine (5-磷酸核糖胺) from PRPP and Gln catalyzed by amido-phosphoribosyl transferase 酰胺磷酸核糖轉(zhuǎn)移酶 The amide group (酰胺基) from Gln displaces the PPi group attached to C-1 of PRPP The configuration at C-1 is inverted from to in this reaction The resulting C-N glycosidic bond has configuration that

8、is characteristic of naturally occurring Nt -構(gòu)型是天然存在的核苷酸中的糖苷鍵的構(gòu)型 This reaction is driven forward by the hydrolysis of PPi PRPPphosphoribosylamine 磷酸核糖胺 glycinamide ribonucleotide 甘氨酰胺核苷酸 formylglycinamide ribonucleotide 甲酰甘氨酰胺核苷酸 formylglycinamidine ribonucleotide 甲酰甘氨脒核苷酸 5-aminoimidazole ribonucle

9、otide 5-氨基咪唑核苷酸 1N 2C2N 3C2N 3C3N成環(huán)(3C2N+側(cè)鏈1N) 胺基1N + Gly 2C1N+甲?；?C+胺基1N 咪唑環(huán)3C2N +側(cè)1N 5-aminoimidazole-4-carboxylate ribonucleotide 5-氨 基咪唑 4-羧酸核苷酸5-aminoimidazole-4-N-succinocarboxamide ribonucleotide 5-氨基咪唑4- N-琥珀酸氨甲酰核苷酸 5-aminoimidazole-4- carboxamide ribonucleotide 5-氨基咪唑 4-氨甲酰核苷酸 5-formamidoim

10、idazole-4-carboxamide ribonucleotide 5- 甲酰胺咪唑-4-氨甲酰核苷酸 IMP 次黃苷酸 (3C2N +) 1N 1C1N 5C2N 1C2N 2C2N成環(huán)(5C4N) 側(cè)鏈氨基1N + CO2 1C +Asp 4C1N 延胡索酸4C+甲酰基1C 次黃嘌呤5C4NAMP and GMP are formed from IMP IMP, the product of the de novo pathway, is the precursor of AMP and GMPIMPadenylosuccinate 腺苷琥珀酸AMPThe difference be

11、tween AMP and IMP is the substitution of an NH2 for the =O at C-6IMPXMP 黃苷酸GMP In the conversions of IMP into AMP and GMP, a carbonyl (羰基) oxygen atom is replaced by an amino group similarly, in the synthesis of IMPstep 4; in the formation of CTP from UTP(P748); and in the conversion of Cit into Arg

12、 in the urea cycle(P635) The common mechanistic theme of these reactions is the conversion of the carbonyl oxygen into a derivative that can be readily displaced by an amino group 羰基氧(含有磷酸基的衍生物)氨基 The reaction mechanism for replacement of a carbonyl oxygen by an amino group is shown by Fig 29-8 The

13、attacking nitrogen can be from NH3, Gln or Asp(三者都有氨基), The leaving group in this class of reactions can be Pi, PPi or the AMP(三者都有磷酸基) moiety Pur bases can be recycled by salvage補(bǔ)救補(bǔ)救 reactions that utilize PRPP Pur Nt (嘌呤核苷酸) can be synthesized from the preformed bases (formed from the hydrolytic d

14、egradation of NA and Nt) by a salvage reaction 節(jié)約利用途經(jīng)(反應(yīng)) which is simpler and much less costly than the reactions of the de novo pathway In the salvage reactions補(bǔ)救途經(jīng)(節(jié)約利用途經(jīng)), the PR moiety of PRPP is transferred to a Pur to form the corresponding Nt Two salvage enzymes with different specificities

15、recover Pur bases Adenine phosphoribosyl transferase 腺嘌呤磷酸核糖轉(zhuǎn)移酶(APRT): Adenine+ PRPPAMP+ PPi Hypoxanthine-guanine phosphoribosyl transferase 次黃嘌呤-鳥(niǎo)嘌呤磷酸核糖轉(zhuǎn)移酶(HGPRT): Hypoxanthine (or guanine)+ PRPPIMP (or GMP)+ PPi. AMP, GMP, and IMP are feedback inhibitors of Pur Nt biosynthesis The synthesis of Pur

16、 Nt is controlled by feedback inhibition and other regulatory mechanisms at several sites 5-phosphoribosyl-1-pyrophosphate synthetase 5- 磷酸核糖-1-焦磷酸合成酶, the enzyme that synthesizes PRPP, is partially inhibited by Pur Nt The enzyme is not totally switched off when Pur are abundant for PRPP is also a p

17、recursor of Pyr and of His The committed step in Pur Nt biosynthesis is the conversion of PRPP into phosphoribosylamine 磷酸核糖胺 by Gln-PRPP amidotransferase 谷酰胺PRPP酰胺基轉(zhuǎn)移酶 This key enzyme is feedback-inhibited by many Pur Nt It is noteworthy that AMP and GMP, the final products of the pathway, are syne

18、rgistic 協(xié)同的in inhibiting the amidotransferase Inosinate (IMP) (次黃苷酸) is the branch point in the synthesis of AMP and GMP The reactions leading away from IMP are sites of feedback inhibition AMP and GMP inhibit the conversion of IMP into adenylosuccinate 腺苷琥珀酸 and IMP into XMP黃苷酸 GTP and ATP are the

19、substrates in the synthesis of AMP and GMP reciprocally 交互 This reciprocal substrate relation tends to balance the synthesis of adenine and guanine Nt 使兩種嘌呤核苷酸的合成平衡 In E.coli, most of the gene encoding enzymes of the de novo pathway are coordinately regulated Specifically, their transcription is blo

20、cked by the purine repressor (Pur R) 嘌呤阻抑蛋白, a DNA-binding protein, when hypoxanthine (次黃嘌呤) and guanine are abundant. 嘌呤嘌呤阻抑蛋白與DNA結(jié)合 阻斷轉(zhuǎn)錄編碼從頭合成嘌呤核苷酸的酶The Pyr ring is synthesized from carbamoyl phosphate 氨甲酰磷酸氨甲酰磷酸and Asp the Pyr ring is assembled first and then linked to PR to form a Pyr Nt, in con

21、trast with the reaction sequence in the de novo synthesis of Pur Nt 嘌呤核苷酸的合成是在磷酸核糖(PR)的基礎(chǔ)上合成嘌呤環(huán)(堿基);而嘧啶核苷酸的合成則是先合成嘧啶環(huán)(堿基),再加入磷酸核糖(PR). The precursors of the Pyr ring are carbamoyl phosphate and Asp There are two major differences in the synthesis of carbamoyl phosphate used to synthesize Pyr and to

22、make urea compartmentation: (in eukaryotes) in the cytosol (for Pyr) and in mito (for urea), respectively, and by different carbamoyl phosphate synthetase 區(qū)域不同(合成酶不同) Gln rather than NH4+ is the N donor in the cytosolic synthesis of carbamoyl phosphate 氮供體不同 Also, N-acetylglutamate does not serve as

23、 an allosteric activator in the cytosolic synthesis. (變構(gòu)激活劑需否) Gln+ 2ATP+ HCO3 carbamoyl phosphate+ 2ADP+ Pi+ Glu The committed step in the biosynthesis of Pyr is the formation of N-carbamoylaspartate N-氨甲酰天冬氨酸 from Asp and carbamoyl phosphate 4C1N + 1C1N = 5C2N = 4C2N(嘧啶環(huán)) +1C(側(cè)鏈羧基,后脫去) This carbam

24、oylation 氨甲?；?is catalyzed by Asp transcarbamoylase 天冬氨酸轉(zhuǎn)氨甲酰酶 The Pyr ring is formed in the carbamoylaspartate cyclization環(huán)化 with loss of water to yield dihydroorotate (DHO, 二氫乳清酸,由二氫乳清酸酶催化). Orotate (乳清酸) is then formed by dehydrogenation of DHO(由二氫乳清酸脫氫酶催化). Orotate acquires a PR moiety from PRPP

25、to form a Pyr Nt Orotateorotidylate (OMP) (乳清苷酸)UMP acquisition of a PR group from PRPP to form a Pyr Nt, this reaction is driven by the hydrolysis of PPi OMP is decarboxylated to yield UMP, a major Pyr Nt Pyr biosynthesis in higher organisms is catalyzed by multifunctional enzymes In E .coli 6 enzy

26、mes that synthesize UMP from simple precursors appear to be unassociated In eukaryotes, by contrast, 5 of them are clustered in two complexes One of these multifunctional enzymes was discovered when cultured mammalian cells were treated with N-(phosphonacetyl)-L-Asp (PALA, N-磷酸乙酰-L-天冬氨酸) 用抑制劑處理培養(yǎng)的哺乳

27、細(xì)胞,發(fā)現(xiàn)(在克服抑制作用而繼續(xù)存活的細(xì)胞中)三種酶(Carbamoyl phosphate synthetase, Asp transcarbamoylase, and DHOase )的濃度都(同時(shí))提高了100倍 Carbamoyl phosphate synthetase, Asp transcarbamoylase, and DHOase are covalently joined in a single 240-kd polypeptide chain This multifunctional enzyme is called CAD Orotate乳清酸PR transferas

28、e and OMP decarboxylase are also associated in eukaryotes Multifunctional enzymes also mediate the synthesis of Pur in vertebrates (step2+3+5; 6+7; 9+10) Indeed, the covalent linkage of functionally related enzymes occurs often in eukaryotes 真核生物中功能相關(guān)的酶經(jīng)常共價(jià)連接在一起(在同一條多肽鏈上) The mammalian FA synthase,

29、which contains 7 enzymatic activities in each of two chains, is another striking example The clustering of enzymes catalyzing a reaction sequence has several potential advantages their synthesis is coordinated and their assembly into a coherent complex is easily assured協(xié)調(diào)合成 side reactions are minimi

30、zed as substrates are channeled from one catalytic site to the next副作用少 a covalently linked multifunctional complex is likely to be more stable than one formed by noncovalent interactions穩(wěn)定 Multifunctional enzymes probably evolved by exon shuffling外顯子改組Nucleoside (Ns) mono-, di-, and triphosphates a

31、re interconvertible The active forms of Nt in biosynthesis and energy conversions are NDP and NTP conversion of NMP to NDP is catalyzed by specific nucleoside monophosphate kinase 核苷單磷酸激酶 (12) that utilize ATP as the phosphoryl donor e.g : UMP+ ATPUDP+ ADP; AMP+ ATP2ADP Ns diphosphates and triphosph

32、ates are interconverted by nucleoside diphosphate kinase 核苷二磷酸激酶, an enzyme that has broad specificity (23) XDP+ YTP XTP + YDP X and Y can be any of several ribonucleosides or deoxyribonucleosidesCTP is formed by aminationan of UTP both CTP and UTP are the major Pyr ribonucleotides The only differen

33、ce: carbonyl oxygen at C-4(UTP) is replaced by an amino group(CTP) In mammals, amide group of Gln is amino donor, whereas in E.coli NH4+ is used in this reaction Mammals avoids having a high level of NH4+ in plasma by generating it in situ 原位from a donor such as Gln ATP is consumed in both amination

34、 reactions As in the conversions of IMP to AMP and GMP, an acyl phosphate intermediate酰基磷酸中間物 is nucleophilically親核 attacked by a nitrogen atom Pyr Nt biosynthesis in bacteria is regulated by feedback inhibition The committed step in Pyr Nt biosynthesis in E.coli is the formation of N-carbamoylaspar

35、tate from Asp and carbamoyl phosphate Aspartate transcarbamoylase (ATCase), the enzyme that catalyzes this reaction is feedback-inhibited by CTP, the final product in the pathway A second control site is carbamoyl phosphate synthetase, which is feedback-inhibited by UMP Ribonucleotide reductase 核苷酸還

36、原酶核苷酸還原酶, a radical自由基自由基 enzyme, catalyzes the synthesis of deoxyribonucleotides脫氧核苷酸脫氧核苷酸 dNt are the precursors of DNA, that are formed by the reduction of ribonucleotides The 2-hydroxyl group on the ribose moiety is replaced by a hydrogen atom substrates are ribonucleoside diphosphates or tripho

37、sphates (NDP or NTP), and ultimate reductant is NADPH: NDPdNDP The electrons from NADPH are transferred to the substrate through a series of carriers: a flavin黃素 the sulfhydryls巰基 of a small protein a pair of irons that generate a tyrosyl radical and then another pair of sulfhydryls Ribonucleotide r

38、eductase catalyzes the final stage: The substrate specificity and catalytic activity of ribonucleotide reductase are precisely controlled There are two allosteric sites in ribonucleotide reductase: one for overall activity(1), another for substrate specificity(2,3,4) 1. NDP dNDP ATP+, dATP (overall

39、catalytic activity) 核糖核苷酸促進(jìn),脫氧核糖核苷酸抑制(脫氧核糖核苷酸的形成), 以保持核糖核苷酸和脫氧核糖核苷酸之間的平衡 2. UDP (CDP) dUDP (dCDP) dATP or ATP+ (the balance between Py and Pu)嘌呤核苷酸(或脫氧嘌呤核糖核苷酸)促進(jìn)脫氧嘧啶核糖核苷酸合成 3. GDPdGDP dTTP+ (the balance between Py and Pu) 脫氧嘧啶核糖核苷酸促進(jìn)脫氧嘌呤核糖核苷酸合成 4. ADPdADP dGTP+ (the balance between two different Pu

40、Nt)鳥(niǎo)嘌呤脫氧核糖核苷酸促進(jìn)腺嘌呤脫氧核糖核苷酸合成Thioredoxin 硫氧還蛋白硫氧還蛋白 and glutaredoxin 谷氧還蛋白谷氧還蛋白carry electrons to ribonucleotide reductase(RR) Two carriers of reducing power to ribonucleotide reductase were foundthioredoxin and glutaredoxin The process of the transfer of the reducing power (NADPH還原力 RR核糖核苷酸還原酶ribose

41、unit底物) is shown: NADPHTR (FAD+TR) 硫氧還蛋白還原酶T硫氧還蛋白R(shí)Rribose unit NADPHGR (GR+ FAD) 谷氧還蛋白還原酶G 谷胱甘肽GX 谷氧還蛋白R(shí)Rribose unit Deoxythymidylate (dTMP) is formed by methylation甲基化甲基化 of deoxyuridylate (dUMP) uracil is not a component of DNA Rather, DNA contains thymine, the methylated analog of uracil thymidyl

42、ate synthase胸苷酸合酶catalyzes this finishing touch: dUMP is methylated to dTMP The methyl donor in this reaction is a FH4 derivative (N5,N10-methylene FH4) rather than S-A Met The methyl group inserted into dTMP is more reduced than the methylene group in the donor What is the source of electrons for t

43、his reduction? The two electrons come in the form of a hydride ion (H) from the FH4 moiety itself This hydrogen es part of the methyl group of dTMP In this reaction, FH4 is oxidized to FH2. Thus N5, N10-methylene FH4 serves both as an electron donor and as a one-carbon donor in the methylation react

44、ion 既作為電子供體,又作為一碳單位供體 We see here, as in the synthesis of Pu, the key role of FH4 derivatives. Indeed, Nt metabolism and AA metabolism are closely tied by one-carbon transfer 核苷酸代謝和AA代謝通過(guò)一碳單位轉(zhuǎn)移而密切連接Three-dimensional structure of E.coli dihydrofolate reductase with a bound methotrexate. It is notewor

45、thy that the deoxyribose and thymine units of DNA are formed by modification of ribonucleotides DNA中的脫氧核糖和胸腺嘧啶由RNA中的核糖核苷酸修飾后形成 In contrast, no known ribonucleotide is formed from a deoxyribonucleotide These precursor-product relations strongly imply that ribonucleotides came first in evolution The r

46、eactions catalyzed by ribonucleotide reductase and thymidylate synthase are recapitulations (重述) of the transition from a RNA world to one in which DNA became the store of genetic information Potent competitive inhibitors of dihydrofolate reductase(A)The anticancer drugs aminopterin氨基蝶呤(4-氨基葉酸) and

47、methotrexate氨甲蝶呤 contain an NH2 group in place of the OH group of dihydrofolate. Methotrexate also differs in having a CH3 group instead of H at N10.(B)Trimethoprim三甲氧芐二氨嘧啶, an antibacterial folate analog. FH2 reductase catalyzes the regeneration of FH4, a one-carbon carrier For carrying OCU, FH4 mu

48、st be regenerated from FH2 that is produced in the synthesis of dTMP This is plished by FH2 reductase using NADPH as the reductant: FH2+NADPH+ H+ FH4+ NADP+ A hydride ion is directly transferred from nicotinamide ring of NADPH to pteridine ring of FH2 Several valuable anticancer drugs block the synt

49、hesis of dTMP Rapidly dividing cells require an abundant supply of dTMP for the synthesis of DNA The vulnerability易受性 of these cells to the inhibition of dTMP synthesis has been exploited in cancer chemotherapy dTMP synthase胸苷酸合酶 and FH2 reductase 二氫葉酸還原酶 are choice target enzymes Fluorouracil 氟尿嘧啶

50、or fluorodeoxyuridine 氟脫氧尿苷, a chemically useful anticancer drug, is converted in vivo into fluorodeoxyuridylate (F-dUMP) This analog of dUMP irreversibly inhibits thymidylate synthase after acting as a normal substrate through part of the catalytic cycle First, a sulfhydryl group of the enzyme adds

51、 to C-6 of the bound F-dUMP -CH2-FH4 then adds to C-5 of this intermediate In the case of dUMP, a hydride ion of the folate is subsequently shifted to the CH2-, and a proton is taken away from C-5 of the bound Nt However, F+ cannot be abstracted from F-dUMP by the enzyme, and so catalysis is blocked

52、 at the stage of the covalent complex formed by (1) F-dUMP, (2) CH2-FH4, and (3)-SH of the enzyme This is an example of suicide inhibition自殺抑制, in which an enzyme converts a substrate into a reactive inhibitor that immediately inactivates its catalytic activity The synthesis of dTMP can also be bloc

53、ked by inhibiting the regeneration of FH4 Analogs of FH2, such as aminopterin氨基蝶呤 and methotrexate 氨甲蝶呤, are potent competitive inhibitors (Ki 1nmol/L) of FH2 reductase Methotrexate is a valuable drug in the treatment of many rapidly growing tumors, such as acute leukemia and choriocarcinoma 絨毛膜癌 Ho

54、wever, it is quite toxic because it kills rapidly replicating cells whether they are malignant惡性 or not Stem cells in bone marrow 骨髓, epithelial cells上皮細(xì)胞of the intestinal tract, and hair follicles毛囊 are vulnerable to the action of this folate antagonist拮抗劑, accounting for many of its toxic side eff

55、ects Folate analogs such as trimethoprim 三甲氧芐二氨嘧啶binds 105-fold less tightly to mammalian FH2 reductase than it does to reductases of susceptible microorganisms NAD+, FAD, and CoA are formed from ATP NAD+: nicotinate 煙酸, 或尼克酸nicotinate Nt煙酸核苷酸 Nicotinate is derived from Trp. Humans can synthesize th

56、e required amount of it if the supply of Trp in the diet is adequate However, an exogenous supply of it is required if the dietary intake of Trp is low A dietary deficiency of Trp and nicotinate can lead to pellagra 糙皮病, a disease charecterized by dermatitis 皮炎, diarrhea 腹瀉, and dementia 癡呆 Nicotina

57、te Nt desamido-NAD+ 脫酰胺NAD+ NAD+ NADP+ is derived from NAD+ by phosphorylation of the 2-hydroxyl group of the adenine ribose moiety This transfer of a phosphoryl group from ATP is catalyzed by NAD+ kinase FAD: riboflavin 核黃素(VB2)riboflavin 5-phosphate (or flavin mononucleotide) flavin adenine dinucl

58、eotide 黃素腺嘌呤二核苷酸 Both 5-phosphate and AMP unit come from ATP, so 2 ATP consumed here CoA: the AMP moiety of CoA also comes from ATP pantothenate泛酸(+ATP) 4-phosphopantothenate 4-磷酸泛酸(+Cys+ATP) 4-phosphopantothenyl cystein 4-磷酸泛酰半胱氨酸(CO2)4-phosphopantotheine 4-磷酸泛酰巰基乙胺(+ATP) dephospho-CoA 脫磷酸CoA(+ATP)

59、CoA A common feature of the biosyntheses of NAD+, FAD, and CoA is the transfer of the AMP moiety of ATP to the phosphate group of a phosphorylated intermediate ATP將AMP(單位)轉(zhuǎn)移到磷酸化中間物的磷酸基上 The PPi formed in these condensations is then hydrolyzed to Pi As in many other biosyntheses, much of the thermody

60、namic driving force comes from the hydrolysis of the released PPi Pu in humans are degraded to urate 尿酸尿酸 The Nt of a cell undergo continuous turnover Nt are hydrolytically degraded to Ns by nucleotidase 核苷酸酶 Phosphorylytic cleavage of Ns to free bases and R-1-P (or deoxyribose 1-P) is catalyzed by