Nucleotides metabolism

【目的与要求】 记住嘌呤核苷酸有两条合成途径。结合嘌呤核苷酸结构与从头合成途径，说出嘌呤核苷酸各元素或组件的材料来源。熟记二磷酸核苷还原生成脱氧嘌呤核苷酸。写出与嘌呤核苷酸补救合成有关的酶的名称、功能、酶缺陷相关的疾病 结合嘌呤核苷酸合成途径、调节，熟记嘌呤核苷酸抗代谢药物作用机理及临床意义 记住嘌呤核苷酸体内分解代谢终产物-尿酸及其与医学的关系 熟记嘧啶核苷酸从头合成的原料及合成调节。说出嘧啶核苷酸补救合成所需的酶及其催化的反应。明白嘧啶核苷酸抗代谢药物作用机理，记住嘧啶核苷酸分解代谢产物名称

Outline 8.1 Purine metabolism -8.1.1 The Biosynthesis of Purines -8.1.2 Purine Salvage -8.1.3 De-oxyribonucleotide Synthesis -8.1.4 Purine Degradation 8.2 Pyrimidine metabolism -8.2.1 Biosynthesis of Pyrimidines -8.2.2 Pyrimidine Degradation

Biological Roles of Nucleotides Monomeric units of nucleic acids * “ Energy currency”(ATP) * Regulation of physiological processes Adenosine controls coronary(冠脉) blood flow cAMP and cGMP serve as signaling molecules Precursor function (GTP to tetrahydrobiopternin) Coenzyme components ( 5’-AMP in FAD/NAD+) Activated intermediates: UDP-Glucose Allosteric effectors- regulate themselves and others

Nuclear acid digestion food (stomach) protein nuclear acid (RNA and DNA) (intestine) RNase (phosphodiesterase) Endonucleases DNase mononucleotide ribonucleotide mmol Deoxyribonucleotide umol (phosphoesterase) Nucleotidase nucleoside Phosphate nucleosidase Uric acid (purines) base Ribose or ribose-1-phosphate β-ureidopropionate ( primidines) (戊糖代谢) excrete

思 考？ 利用所学知识，试评价“珍奥核酸”的功能

8.1.1 Nucleotide Biosynthesis For both purines and pyrimidines there are two means of synthesis - de novo (from bits and parts) - salvage (recycle from pre-existing nucleosides,and bases) Ribose generates energy, but purine and pyrimidine rings do not Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies

Bases/Nucleosides/Nucleotides Base + Sugar + Phosphate= Deoxyadenosine 5’-triphosphate (dATP) Adenine Deoxyadenosine

The Pyrimidine Ring The Purine Ring

Purine C N HC H CH NH2 腺嘌呤A O C N H CH 6 H2N 2 鸟嘌呤G

Pyrimidine C HN CH N H CH3 O Thymine NH2 O C HN CH N H Cytosine

De novo purine biosynthesis John Buchanan (1948) "traced" the sources of all nine atoms of purine ring 1. In de novo synthesis, Inosine-5'-P (Inosine Monophosphate, IMP) is the first nucleotide formed 2. It is ,then, converted to either AMP or GMP Location: liver cellular Cytoplasm De novo purinenucleotide synthesis proceeds by the synthesis of the purine base upon the ribose sugar moiety

The metabolic origin of the nine atoms in the purine ring system N-1: aspartic acid C-2:THF - one carbon units N-3: glutamine C-4, C-5, N-7: glycine C-6: CO2 C-8: THF - one carbon units N-9: glutamine 甘氨当中站, 谷氮坐两边, 左上天冬氨, 头顶CO2 二八俩一碳 C-6 N-7 C-4 C-5 N-1 C-8 C-2 N-9 H N-3 The metabolic origin of the nine atoms in the purine ring system

1. First, synthesis Inosine-5'-P (Inosine Monophosphate, IMP)

R-5'-P ATP PRPP synthetase P_ P PP-1'-R-5'-P(PRPP)

Figure 22-31-01 5-磷酸核糖胺,PRA T1/2 30s

Figure 22-31-02 甘氨酰胺核苷酸(GAR)

Figure 22-31-03 甲酰甘氨酰胺核苷酸 (FGAR)

Figure 22-31-04 甲酰甘氨咪核苷酸(FGAM)

Figure 22-31-05 5-氨基咪唑核苷酸 (AIR)

Figure 22-31-06 5-氨基咪唑-4-羧酸核苷酸

5-氨基咪唑-4-(N-琥珀酸) -甲酰胺核苷酸(SAICAR) Figure 22-31-07 5-氨基咪唑-4-(N-琥珀酸) -甲酰胺核苷酸(SAICAR)

5-氨基咪唑-4-(N-琥珀酸) -甲酰胺核苷酸(SAICAR) Figure 22-31-08 5-氨基咪唑-4-(N-琥珀酸) -甲酰胺核苷酸(SAICAR)

5-氨基咪唑-4-甲酰胺 核苷酸(AICAR) Figure 22-31-09 5-氨基咪唑-4-甲酰胺 核苷酸(AICAR)

5-甲酰胺基咪唑- 4-甲酰胺核苷酸(FAICAR) Figure 22-31-10 5-甲酰胺基咪唑- 4-甲酰胺核苷酸(FAICAR)

Figure 22-31-11

Inosine monophosphate (2) ATP dependent step ATP dependent step PRPP NH3 via glutamine Inosine monophosphate 1 carbon via folate NH3 via aspartyl- succinate

2.Second, Making AMP and GMP

ATP AMP ADP GTP GMP GDP kinase kinase kinase kinase ATP ADP ATP ADP

Purines are synthesized on the Ribose ring Committed Steps ( at the first two steps ): PRPP , PRA (A bunch of steps you don’t need to know) 2.End product inhibition and “feed forward” regulation Regulation of De Novo Synthesis 3. “cross regulation” occurs from IMP to AMP and GMP ATP provides the energy for GMP synthesis GTP provides the energy for AMP synthesis Feedback Inhibition

Committed Step

8.1.2 Salvage Pathway for Purines Hypoxanthine or Guanine + PRPP = IMP or GMP + PPi Hypoxanthineguanosylphosphoribosyl transferase (HGPRTase) Adenine + PRPP = AMP + PPi Adeninephosphoribosyl transferase (APRTase) A-PRT(phosphoribosyltransferases) is not very important because we generate very little adenine. (the catabolism of adenine nucleotides and nucleosides is through inosine) HG-PRT, though, is exceptionally important and it is inhibited by both IMP and GMP. This enzyme salvages guanine directly and adenine indirectly. Remember that AMP is generated primarily from IMP, not from free adenine PRTs catalyze the addition of ribose 5-phosphate to the base from PRPP to yield a nucleotide Base + PRPP = Base-ribose-phosphate + PPi Salvage pathways are particularly important in brain/marrow that lack de novo purine synthesis

Lesch-Nyhan Syndrome(莱-尼综合症) Absence of HGPRTase X-linked (Gene on X) Occurs primarily in males Characterized by: purine synthesis is increased 200-fold Increased uric acid Spasticity(痉挛) Neurological defects Aggressive behavior Self-mutilation(自残)

Inter-conversion of Purine nucleotides NADP+ NH3 NADPH Guanine Reductase GMP AMP NH3 Adenine Deaminase AMPS （腺苷酸代琥珀酸） IMP XMP

8.1.3 Deoxyribonucleotide Biosynthesis 1´ 2´ 3´ 4´ 5´ BASE 1´ 2´ 3´ 4´ 5´ BASE Ribonucleotide Reductase Deoxyribonucleoside Ribonucleoside

ADP dADP GDP dGDP UDP dUDP CDP dCDP TDP dTDP ribonucleotide reductase ADP dADP ribonucleotide reductase GDP dGDP ribonucleotide reductase UDP dUDP ribonucleotide reductase CDP dCDP TDP dTDP

Deoxyribonucleotide Biosynthesis ? 硫氧还蛋白 Mg2+ Ribonucleotides can be converted to deoxyribonucleotides by Ribonucleotide Reductase at the diphosphate level

Regulates the level of cellular dNTPs E. coli Ribonucleotide Reductase Regulates the level of cellular dNTPs Highly regulated enzyme Activated prior to DNA synthesis Controlled by feedback inhibition by dATP, and complex positive regulation by TTP, dGTP and dGTP The ribonucleotide reductase, An (R1)2(R2)2- type enzyme , has R1 (86 kD) and R2 (43.5 kD) two subunits

? kinase dADP+ATP dATP +ADP kinase dGDP+ATP dGTP+ADP kinase dUDP+ATP dUTP+ADP kinase dCTP+ADP dCDP+ATP phosphorylase dNDP dNMP+Pi ? dTTP

Regulation of dNTP Synthesis The overall activity of ribonucleotide reductase must be regulated Balance of the four deoxynucleotides must be controlled ATP activates, dATP inhibits at the overall activity site ATP, dATP, dTTP and dGTP bind at the specificity site to regulate the selection of substrates and the products made

Regulation of dNTP Synthesis Figure 22-40

over-growth + Heterogeneity ( nucleotides + protein ) Tumor over-growth + Heterogeneity ( nucleotides + protein ) How to inhibit the biosynthesis of the tumor cells? for anti-cancer strategies(antibacterial)

Chemotherapeutic Agents 1. Analogs of purine: N OH H N SH H 6-巯基嘌呤 （6-mercaptopurine, 6-MP） inosine N SH H H2N N OH H 8-氮杂鸟嘌呤 (8-azoguanine) 6-巯基鸟嘌呤 (6-mercaptoguanine)

2. Analogs of amino acids: H2N—C—CH2—CH2—CH—COOH O NH2 Gln Inhibit the reactions of the Gln N+ —N—CH2—C—O—CH2—CH—COOH O NH2 氮杂丝氨酸（azaserine） N+ —N—CH2—C—CH2—CH2—CH—COOH O NH2 6-重氮-5-氧正亮氨酸(diazonnorleucine)

3. Analogs of Folic acid NH2 R O N —CH2—N— —C—N—CH CH2 H2N COOH R=H，aminopterin,氨喋呤 R=CH3，methotrexate, 氨甲喋呤,MTX N C H —CH2—N— —C—N—CH O CH2 COOH H2N OH 四氢叶酸,FH4

The mechanism of the Chemotherapeutic Agents 6MP MTX PRPP PRA GAR FGAR Gln azaserine 氮杂丝氨酸(azaserine) FGAM PPi PRPP 6MP AMP A MTX PPi PRPP AICAR FAICAR IMP I GMP G 6MP azaserine PPi PRPP The mechanism of the Chemotherapeutic Agents 6MP

8.1.4 Purine catabolism AMP I Uric Acid X GMP G XO XO Sequential removal of bits and pieces End product is uric acid XO: Xanthine Oxidase Excreted in Urine

Xanthine Oxidase and Gout ＞0.48mmol/L(8mg%), The scale of uric acid (normal value) : 0.12～0.36mmol/L; male, 0.27mmol/L; formale, 0.21mmol/L XO in liver, intestines (and milk) can oxidize hypoxanthine (twice) to uric acid Humans and other primates(灵长类) excrete uric acid in the urine, but most N goes out as urea Gout occurs from accumulation of uric acid crystals in the extremities

Allopurinol, which inhibits XO, is a treatment of gout 别嘌呤醇 次黄嘌呤 I

Allopurinol nucleotide The mechanism of allopurinol as a treatment of gout C OH N H N OH H CH allopurinol PRPP ↓ I XO Allopurinol nucleotide X Purine nucleotides Uric acids

8.2 Pyrimidine Biosynthesis In contrast to purines, First, synthesis of the pyrimidine ring; Next, attachment of ribose-phosphate to the ring

The metabolic origin of the six atoms of the pyrimidine ring De Novo Pyrimidine Biosynthesis NH2 HC H2C HOOC Carbamoyl-P Aspartate The metabolic origin of the six atoms of the pyrimidine ring

乳清酸 二氢乳清酸 乳清酸核苷酸

UTP UMP UDP kinase kinase CTP From UTP at the triphosphate level ATP ADP ATP ADP

Synthesis of Thymine Nucleotides 1. Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP 2. Biosynthesis of deoxyribonucleotides by ribonucleotide reductase Figure 22-41 3. Biosynthesis of thymidine monophosphate (dTMP) by thymidylate synthase

dUMP dTMP dTTP dTMP dTDP Thymidylate synthase methylates dUMP at 5-position to make dTMP C O HN CH N dR-5'-P C O HN C-CH3 CH N dR-5'-P dTMP synthase N5,N10- methylene FH4 FH2 DHFR FH4 NADP+ NADPH+H+ dUMP dTMP N5,N10-methylene THF is 1-C donor kinase kinase dTTP dTMP dTDP ATP ADP ATP ADP

Regulation of Pyrimidine Synthesis（de novo） Aspartate transcarbamoylase (ATCase 细菌) catalyzes the condensation of carbamoyl phosphate with aspartate to form carbamoyl-aspartate Note that carbamoyl phosphate represents an ‘activated’ carbamoyl group Feedback Inhibition

× Feedback Inhibition Regulation of Pyrimidines Biosynthesis Regulation occurs at first step in the pathway (committed step) Inhibited by UTP If you have lots of UTP around this means you won’t make more that you don’t need Feedback Inhibition × 2ATP + CO2 + Glutamine = carbamoyl phosphate

CPS II Carbamoyl phosphate for pyrimidine synthesis is made by carbamoyl phosphate synthetase II (CPS II 哺乳动物细胞) This is a cytosolic enzyme (whereas CPS I is mitochondrial and used for the urea cycle) Substrates are HCO3-, glutamine, 2 ATP

Allosteric regulation of pyrimidine biosynthesis Enzyme regulated Allosteric effector Effect carbamoyl phosphate synthetase II UDP, UTP Feedback inhibition PRPP, ATP stimulatory

CPS-I vs. CPS-II ?

Biosynthesis: Purine vs. Pyrimidine start with ribose, build on nitrogen base Regulated by GTP/ATP Generates IMP Requires Energy build nitrogen base then added to PRPP Synthesized Regulated by UTP Generates UMP/CMP Requires Energy “Both are very complicated multi-step process which your kindly professor does not expect you to know in detail”

Salvaging Pyrimidines Pyrimidines+PRPP  Nucleoside+PPi (嘧啶磷酸核糖转移酶) A second type of salvage pathway involves two steps and is the major pathway for the pyrimidines, uracil and thymine Base + Ribose 1-phosphate = Nucleoside + Pi (nucleoside phosphorylase) Nucleoside + ATP  Nucleotide + ADP (nucleoside kinase-irreversible) Uridine + ATP  UMP + ADP; uridine kinase Nucleoside phosphorylase/kinase: U,C,T + ribose 1-P  nucleosides + Pi Thymine + deoxyribose-1-P  thymidine + Pi; thymine phosphorylase Thymine + ATP  dTMP + ADP; thymidine kinase Thymidine kinase activity changes during cell cycle; very active during DNA synthesis and is inhibited by dTTP

Analogs of pymidines /pymidine nucleosides: Inhibitors of pymidines synthesis are cancer drugs Analogs of pymidines /pymidine nucleosides: HOH2C H OH O C N NH·HCl HOH2C H HO OH O C N NH2 N H O F 5-氟尿嘧啶 5-Fu 环胞苷 Cyclocytidine 阿糖胞苷 Cytarabine

UMP UTP CTP CDP dCDP MTX dTMP UDP dUDP dUMP 5FU 氮杂丝氨酸 azaserine 阿糖胞苷Cytarabine UMP UTP CTP CDP dCDP MTX dTMP UDP dUDP dUMP 5FU (5FdUMP/5FUTP)

Pyrimidine Catabolism-1 CH NH2 H C O HN CH N H C O HN CH2 N H NADPH+H+ NADP+ NH3 H2O C U C O H2N CH2 N H HO CO2+NH3 β-Alanine H2O H2N-CH2-CH2-COOH

Pyrimidine Catabolism-2 HN C-CH3 CH N H C O HN CH-CH3 CH2 N H NADPH+H+ NADP+ DHT T H2O Pyrimidine Catabolism-2 C O H2N CH-CH3 CH2 N H HO CO2+NH3 H2O H2N-CH2-CH-COOH CH3 β-氨基异丁酸 β-aminoisobutyrate β-脲基异丁酸

overview De novo synthesis 5'-P-R CO2+Gln PRPP H2N-CO-P IMP OMP AMP dAMP dGMP GMP UMP dUMP CMP dCMP dTMP ADP dADP dGDP GDP UDP dUDP dUDP CDP dCDP dTDP dUTP ATP dATP dGTP GTP UTP CTP dCTP dTTP De novo synthesis

CPS-I vs. CPS-II 分布 线粒体(肝) 胞液 氮源 氨 谷氨酰胺 变构激活剂 N-乙酰谷氨酸 无 变构抑制剂 无 UMP 功能 尿素合成 嘧啶合成

选择题练习 核苷酸代谢

嘧啶核苷酸生物合成途径的反馈抑制是由于控制了下列哪种酶的活性 ? A. 二氢乳清酸酶 B. 乳清酸磷酸核糖转移酶 C. 二氢乳清酸脱氢酶 D. 天冬氨酸转氨甲酰酶 E. 胸苷酸合成酶

2. 5-氟尿嘧啶的抗癌作用机理是 A. 合成错误的DNA B. 抑制尿嘧啶的合成 C. 抑制胞嘧啶的合成 D. 抑制胸苷酸的合成 E. 抑制二氢叶酸还原酶

3. 哺乳类动物体内直接催化尿酸生成的酶是 A. 尿酸氧化酶 B. 黄嘌呤氧化酶 C. 腺苷脱氨酸 D. 鸟嘌呤脱氨酶 E. 核苷酸酶

4. 最能直接联系核苷酸合成与糖代谢的物质是 A. 葡萄糖 B. 6-磷酸葡萄糖 C. 1-磷酸葡萄糖 D. 1,6-二磷酸葡萄糖 E. 5-磷酸核糖

5. 体內的脱氧核苷酸是由下列哪类物质直接还原而成的 A. 核糖 B. 核糖核苷 C. 一磷酸核苷 D. 二磷酸核苷 E. 三磷酸核苷

6. 氮杂丝氨酸干扰核苷酸合成, 因为它是下列哪 种化合物的类似物 ? A. 丝氨酸 B. 甘氨酸 C. 天冬氨酸 D. 谷氨酰胺 E. 天冬酰胺

7. 能在体内分解产生-氨基异丁酸的核苷酸是 CMP AMP TMP UMP IMP

关于天冬氨酸氨基甲酰基转移酶的下列说法, 哪一种是错误的? GTP是其反馈抑制剂 是嘧啶核苷酸从头合成的调节酶 是由多个亚基组成 是变构酶 服从米-曼氏方程

9. PRPP酰胺转移酶活性过高可以导致痛风症, 此酶催化 A. 从R-5-P生成PRPP B. 从苷氨酸合成嘧啶环 C. 从PRPP生成磷酸核糖胺 D. 从IMP合成AMP E. 从IMP生成GMP

10. 嘧啶核苷酸从头合成的特点是 A. 在5-磷酸核糖上合成碱基 B. 由FH4提供一碳单位 C. 先合成氨基甲酰磷酸 D. 甘氨酸完整地参入 E. 谷氨酸提供氮原子

11. The main tissue of de nove synthesis of purine nucleotide in vivo is A. thymus gland B. villous coat of small intestine C. liver D. spleen E. marrow

12. The main end product of purine nucleotide katabolic metabolism in human body is A. urea B. creatine C. Creatinine D. uric acid E. -alanine

13. The methyl of thymine come from N10-CHO FH4 N5,N10=CH-FH4 N5,N10-CH2-FH4 N5-CH3FH4 N5-CH=NHFH4

14. 6-mercapto-purine nucleotide doesn’t suppress A. IMPAMP B. IMPGMP C. PRPP amide transferase D. Purine phosphoribosyltransferase E. Pyrimidine phosphoribosyltransferase

15. 嘌呤核苷酸从头合成的原料包括 A 磷酸核糖 B CO2 C 一碳单位 D 谷氨酰胺 E 天冬氨酸

16. PRPP参与的代谢途径有 A 嘌呤核苷酸的从头合成 B 嘧啶核苷酸的从头合成 C 嘌呤核苷酸的补救合成 D NMPNDPNTP

17. 嘧啶核苷酸合成反馈抑制的酶 A 氨基甲酰磷酸合成酶Ⅱ B 二氢乳清酸酶 C 天冬氨酸氨基甲酰转移酶 D 乳清酸核苷酸脱羧酶

18. 叶酸类似物抑制的反应有 A 嘌呤核苷酸的从头合成 B 嘌呤核苷酸的补救合成 C 胸腺嘧啶核苷酸的生成

19. The compound which can produce feedback suppression of purine nucleotide synthesis is A IMP B AMP C GMP D uric acid

20. Which compound produce uric acid as its decomposed metabolism end product ? A AMP B UMP C IMP D TMP