Nucleotides metabolism Enmin Li

论述题-1 请从一位未来医生的角度，围绕高尿酸血症引发痛风及其生化作用机制，论述人类饮食平衡的重要性。

论述题-2 结合临床应用，举例论述各类抗核苷酸代谢物的作用原理。

Goutiness

Digestion, Ingestion and Degradation of Nuclear acids and Nucleotides and Nucleosides and Bases in Food

Bases/Nucleosides/Nucleotides Deoxyadenosine 5’-triphosphate (dATP) Adenine Deoxyadenosine apostrophe

Small intestine epithelial cells Nucleoproteins, RNA/DNA, Nucleotides, Nucleosides and Bases in Food In stomach Gastric acid and pepsin RNA/DNA, Nucleotides, Nucleosides and Bases In small intestine Endonucleases: RNase and DNase Nucleotides, Nucleosides and Bases Ingestion Small intestine epithelial cells Notes * Nucleotides: AMP, GMP, UMP, CMP, TMP, mmol dAMP, dGMP, dCMP, dTMP, mol * Nucleosides: Adenosine, Guanosine, Cytidine, Uridine, Deoxyadenosine, Deoxyguanosine, Deoxycytidine, Thymidine * Bases: Adenine, Guanine, Cytosine, Thymine, Uracil

In small intestine cells, liver cells and kidney cells Uric acid In small intestine cells, liver cells and kidney cells Nucleotidase Oxidase Deaminase AMP GMP IMP Adenosine Inosine Xanthine Guanine Hypoxanthine Guanosine Nucleosidase H2O P NH4+ Adenine ? R-1-P H2O+O2 H2O2 NADP + + NH3 NADPH+H+ GMP reductase

β-aminoisobutyric acid β-uraminoisobutyric acid β-alanine In small intestine cells, liver cells and kidney cells UMP CMP/dCMP Uracil Cytosine Thymine dTMP Nucleotidase Nucleosidase β-aminoisobutyric acid Dihydrouracil Dihydrothymine β-uraminopropion Reductase β-uraminoisobutyric acid Dihydrothyminase Dihydrouracilase β-uraminopropionase β-uraminoisobutyric acidase

Tricarboxylic acid cycle Uric acid Out of body In kidney β-alanine β-aminoisobutyric acid Succinyl CoA Tricarboxylic acid cycle Pantothenic acid Coenzyme A

The fate of uric acid in the various animals Adenine Hypoxanthine Xanthine Guanine 1 2 3 4 Allantoic acid Glyoxylic acid Uric acid Allantoin Urea Notes: Excreted by 1 Primates, Birds, Reptiles, Insects. 2 Other mammals. 3 Teleost fish. 4 Cartilaginous fish and amphibia 5 Marine invertebrates 5 4NH3 + 2CO2

Uric acid  Gout, Urate crystallization The uric acid and the gout Hypoxanthine Xanthine Out of body Hypoxanthine Allopurinol In urine Uric acid  Over 0.48 mmol/L, In the plasma Diabetese nephrosis …… Gout, Urate crystallization in joints, soft tissue, cartilage and kidney

AMP deaminase and diabetes Jenkins RL, McDaniel HG, Atkins L. Changes in AMP deaminase activities in the hearts of diabetic rats. Biochim Biophys Acta. 1991 Apr 29;1077(3):379-84 Department of Biology, Samford University, Birmingham, AL 35229. AMP deaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantitated by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile change to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP Km = 1.78, stimulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria AMP Km = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP Km = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP Km = 0.97, inhibited by ATP, NAD(P). deaminase AMP IMP Uric acid

Nucleotides Biosynthesis Purine Adenine Guanine Pyrimidine Cytosine 1-3 Uracil Thymine de novo pathway salvage pathway

Phosphoribosyl thansferase Salvage nucleotide biosynthesis pathway Adenine, Guanine, Hypoxanthine, Thymine, Uracil Bases Phosphoribosyl thansferase (APRT, HGPRT) PRPP ATP Nucleosides kinase Adenosine, Guanosine, Cytidine, Uridine, or Deoxy…, Thymidine

Lesch-Nyhan Syndrome (Occurs primarily in males) purine synthesis is about increased 200-fold Increased uric acid Spasticity(痉挛) Neurological defects Aggressive behavior Self-mutilation(自残) The metabolic consequences of congenital HGPRT deficiency in Lesch-Nyhan syndrome:

Hypoxanthine-guanine phosphoribosyl transferase on X chromosome Loss of HGPRT leads to elevated PRPP levels and stimulation of de novo purine synthesis. One ultimate consequence is increased production of uric acid.

De novo nucleotide biosynthesis pathway

The main bases on nucleotides or nucleosides in the tautomeric forms predominant at pH7.

The metabolic origin of the six atoms in the pyrimidine ring Carbamyl-P Aspartate

UTP and CTP biosynthesis UDP ADP UTP ATP UMP kinase

http://www. ncbi. nlm. nih. gov/entrez/viewer. fcgi http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&id=3228248 _ + UDP ATP CTP PRPP UTP The regulatory circuits that control pyrimidine synthesis in E.coli and animals.

First, synthesis Inosine-5'-Monophosphate, IMP 甘氨右中站 谷氮坐两边 左上天冬氨 头顶二氧碳 二八俩叶酸 Glycine Aspartate N10-formyl-THF N10-formyl-THF Glutamine (amide-N) The metabolic origin of the nine atoms in the purine ring First, synthesis Inosine-5'-Monophosphate, IMP

OH ATP AMP Gln:PRPP amidotransferase 5-磷酸核糖胺,PRA

甘氨酰胺核苷酸

甲酰甘氨酰胺核苷酸

甲酰甘氨咪核苷酸

5-氨基咪唑核苷酸

5-氨基-4-羧基咪唑核苷酸 Carboxyaminoimidazole ribonucleotide (CAIR)

Carboxyaminoimidazole ribonucleotide (CAIR) 7 5-氨基-4-(N-琥珀酸) -甲酰胺咪唑核苷酸

8 5-氨基-4-甲酰胺咪唑核苷酸

9 5-甲酰胺基-4-甲酰胺咪唑核苷酸

10

Second, Making AMP and GMP

ADP, ATP, GDP and GTP biosynthesis kinase kinase AMP ADP ATP GTP GDP GTP GDP kinase kinase GMP GDP GTP ATP ADP ATP ADP

5-Phosphoribosylamine Regulation of De Novo Synthesis of Purine Nucleotides Gln:PRPP amidotransferase Ribose-5-phosphate PRPP 5-Phosphoribosylamine IMP XMP AMP ADP ATP GMP GDP GTP Adenylosuccinate + Synthesase Pyrophosphokinase dehydrogenase 2 Active Inactive Key Step

Interconversion of Purine nucleotides XMP AMPS （腺苷一磷酸代琥珀酸） IMP NH3 Adenine Deaminase NADP+＋ NADPH Guanine Reductase AMP GMP

Deoxyribonucleotide Biosynthesis NADPH Adenine Guanine Cytosine Uracil Phosphorylase dNMP dNTP Kinase DNA Thymine × dTTP ?

Deoxythymidine kinase dTMP dTDP dTTP Deoxythymidine ATP ADP Deoxythymidine kinase dUMP dUDP dCMP dCDP N5,N10-methylene-tetrahydrofolic Acid dTMP synthetase

Ribonucleotides can be converted to deoxyribonucleotides by ribonucleotide reductase The (-S-S-)/(-SH HS-) oxidation-reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase and NADPH. 58-kD 12-kD 86-kD2 43.5-kD 2

GSSG 2GSH NDP Reductase Glutaredoxin 2SH

Allosteric regulation 225 462 Allosteric regulation Ribonucleotide reductase

The free radical mechanism of ribonucleotide reduction  unit substrate  unit

Ribonucleoside diphosphate reductase 1 subunit alpha http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&id=2507304 1 mnqnllvtkr dgsterinld kihrvldwaa eglhnvsisq velrshiqfy dgiktsdihe 61 tiikaaadli srdapdyqyl aarlaifhlr kkaygqfepp alydhvvkmv emgkydnhll 121 edyteeefkq mdtfidhdrd mtfsyaavkq legkylvqnr vtgeiyesaq flyilvaacl 181 fsnypretrl qyvkrfydav stfkislptp imsgvrtptr qfsscvliec gdsldsinat 241 ssaivkyvsq ragiginagr iralgspirg geafhtgcip fykhfqtavk scsqggvrgg 301 aatlfypmwh levesllvlk nnrgvegnrv rhmdygvqin klmytrllkg editlfspsd 361 vpglydaffa dqeeferlyt kyekddsirk qrvkavelfs lmmqerastg riyiqnvdhc 421 nthspfdpai apvrqsnlcl eialptkpln dvndengeia lctlsafnlg ainnldelee 481 lailavrald alldyqdypi paakrgamgr rtlgigvinf ayylakhgkr ysdgsannlt 541 hktfeaiqyy llkasnelak eqgacpwfne ttyakgilpi dtykkdldti aneplhydwe 601 alresikthg lrnstlsalm psetssqisn atngiepprg yvsikaskdg ilrqvvpdye 661 hlhdayellw empgndgylq lvgimqkfid qsisantnyd psrfpsgkvp mqqllkdllt 721 aykfgvktly yqntrdgaed aqddlvpsiq ddgcesgack i 225 439 462 Ribonucleoside diphosphate reductase 1 subunit alpha

Regulation of dNTP Synthesis

Allosteric regulation of nucleotide reductase Substrates Products Activators Inhibitors ADP dATP dGTP GDP dTTP CDP dCTP ATP dATP, dGTP, dTTP UDP dATP, dGTP

Chemotherapeutic Agents for cancers 1. Analogs of purine N OH H SH H2N inosine 6-mercaptopurine 8-azoguanine 6-mercaptoguanine guanine

2. Analogs of amino acids O NH2 H2N—C—CH2—CH2—CH—COOH N+ —N—CH2—C—O—CH2—CH—COOH N+ —N—CH2—C—CH2—CH2—CH—COOH Glutamine, Gln Azaserine（重氮乙酰丝氨酸） Diazonorleucine (6-重氮-5-氧正亮氨酸)

3. Analogs of Folic acid N5,N10-methylene-FH4 (N5,N10-亚甲基四氢叶酸) Aminopterin （氨喋呤） R=H，Methotrexate（氨甲喋呤，MTX）R=CH3 N5,N10-methylene-FH4 (N5,N10-亚甲基四氢叶酸)

The mechanism of the Chemotherapeutic Agents-1 Azaserine（重氮乙酰丝氨酸） 6-MP（6巯基嘌呤） 6MP MTX Azaserine MTX（氨甲蝶呤） PRPP PRA GAR FGAR FGAM AICAR Gln MTX Azaserine FAICAR AMP A PPi PRPP 6MP 6MP IMP I 6MP PPi Azaserine 6MP PRPP GMP G The mechanism of the Chemotherapeutic Agents-1

Analogs of pymidines/pymidine nucleosides 5-FU Cytarabine （阿糖胞苷） Cyclocytidine （环胞苷） N H O F Inhibitors of pymidines synthesis are cancer drugs CH3 Cytosine Thymine Uracil NH2 H2C HO OH C HOH2C NH·HCl

The mechanism of the Chemotherapeutic Agents-2 Azaserine（氮杂丝氨酸） Cytarabine（阿糖胞苷） UMP UTP CTP CDP dCDP MTX（氨甲蝶呤） UDP dUDP dUMP dTMP 5FU (5FdUMP/5FUTP) The mechanism of the Chemotherapeutic Agents-2

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苷

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 supreme 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