1、淮南八公山淮南八公山豆腐制作 Department of Biochemistry, University of Colorado(2002-2007) Derivation of iPS cells from various cells -many species -many tissues Histone acetyltransferase and cancer control Protein degradation, Amino Acid Metablism Biosynthesis of Amino Acids Nitogen Fixation Biosynthesis and Deg

2、radation of Nucleotides Metabolism Regulation Lehninger Principles of Biochemistry, 4th edition Textbook of biochemistry with clinical correlations The significance of amino acid 1. cells / tissues growing, renewing and mending C R H NH3 COO Protein Degradation/ Amino Acid Metablism The significance

3、 of amino acid 2. Take part in important physiological activities 3. Oxidation and supply energy 4. Nitrogen balance Before we get on amino acid metablism: 1. Breakdown of dietary proteins 2. Protein degradation in cells Protein breakdown starts at stomach Optimal pH1.52.5， Low specificity Produce s

4、mall peptide and free amino acids pepsinogen 胃蛋白酶原胃蛋白酶原 + Small Peptide HCl、Pepsin Pepsin Breakdown of dietary proteins Cholecystokinin ( 縮膽囊素縮膽囊素) stimulates secretion of several pancreatic enzymes (胰腺酶）胰腺酶）with activity optima at pH 7 to 8. Pancreas exocrine cells : Trypsinogen(胰蛋白酶原）胰蛋白酶原）, Chymo

5、trypsinogen（糜蛋白（糜蛋白 酶原）酶原）, Procarboxypeptidases （羧羧 肽酶）肽酶）A, B The digestion of proteins in the small intestine（小腸）（小腸）. trypsin chymotrypsinogen Elastase彈性蛋白酶彈性蛋白酶 procarboxypeptidase trypsinogen Enterokinase 腸激酶腸激酶 Chymotrypsin糜蛋白酶糜蛋白酶 Proelastase Carboxypeptidase 羧肽酶羧肽酶 Enterokinase腸激酶腸激酶 :produ

6、ced by cells of the duodenum （十二指腸）（十二指腸） 腸激酶，腸激酶，EK， 酶切位點(diǎn)酶切位點(diǎn) DDDDK Synthesis of the enzymes as inactive precursors: 1 Protects the exocrine cells from destructive proteolytic attack. 2 Transportation 3 Regulation Elaborate mechanism for getting active digestive enzymes. Protein Digestion Proteolyt

7、ic enzymes of pancreatic juice Endopeptidases 內(nèi)肽酶內(nèi)肽酶 Exopeptidases 外切酶外切酶 Trypsin : Arg, Lys (C) Chymotrypsin : Tyr, Trp, Phe, Met, Leu (C) Elastase: Ala, Gly, Ser (C) carboxypeptidase aminopeptidase Cleavage sites and substrates of proteases H2N-CH-C-NH-CH- R2 R1RnRRn-1 OOO amino peptidaseendopepti

8、dase carboxy peptidase amino acid+ H2N-CH-C-NH-CH-COOH RR O dipeptidase amino acid polypeptide dipeptide NH-CH-C-NH-C- NH-CH-C-NH-CH-COOH Cleavage sites and substrates of proteases 一 They hydrolyze the peptides very efficiently. In humans, most globular proteins from animal sources are almost comple

9、tely hydrolyzed to amino acids in the gastrointestinal tract, but some fibrous proteins,such as keratin(角蛋白）角蛋白）, are only partly digested. In addition, the protein content of some plant foods is protected against breakdown by indigestible cellulose husks（外殼）（外殼）. Absorption of free amino acids Tryp

10、sin and Protease in Cell Culture Trypsin和蛋白和蛋白/肽的質(zhì)譜測(cè)序肽的質(zhì)譜測(cè)序 基于基于MS/MS的蛋白質(zhì)序列的蛋白質(zhì)序列 和修飾鑒定和修飾鑒定 Liquid chromatography-coupled mass spectrometery(LC-MS) Tryspin digestion A disease caused by obstruction of the normal pathway by which pancreatic secretions enter the intestine. The enzymogens of the prote

11、olytic enzymes are converted to their catalytically active forms prematurely, inside the pancreatic cells, and attack the pancreatic tissue itself. This causes excruciating pain and damage to the organ that can prove fatal. Acute pancreatitis (急性胰腺炎） and Protease 問(wèn)題：?jiǎn)栴}： 1 1 蛋白酶水解的是什么化學(xué)鍵？蛋白酶水解的是什么化學(xué)鍵

12、？ 2 2 人體消化道內(nèi)有哪些蛋白酶人體消化道內(nèi)有哪些蛋白酶? ? 3 3 酶以酶原的形式存在有什么意義？酶以酶原的形式存在有什么意義？ 思考題：思考題： 有哪些方式將蛋白質(zhì)轉(zhuǎn)變?yōu)榘被幔坑心男┓绞綄⒌鞍踪|(zhì)轉(zhuǎn)變?yōu)榘被幔?Two major systems for the turnover of cellular proteins Lysosomes and Ubiquitin-mediated protein degradation Protein Degradation in Cell Lysosomal Degradation Pathway Proteins A. Endocytose

13、d B. Cytosolic C. Organelles by autophagy Lysosomal hydrolases exhibit acidic pH optimal 溶酶體貯積病溶酶體貯積病 Ubiquitin pathway Ubiquitin ProteasomeE3 ligase (Cullin-RING complex) Protein Degradation System in Cell What is Ubiquitin? Human Ub: MQIFKVTLTGKTITLEVEPNDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKE

14、STLHLVLRLRGG Yeast Ub: MQIFKVTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG Ubiquitylation -Sub Ubiquitin pathway E1 -S-Ub E2 -S-Ub E3 substrates Ub Mono-ubiquitination Change protein conformation, Localization, interaction Polyubiquitination Ub Ub Ub Ub substrates Proteosome

15、 Degradation E1- Ub activating enzyme(Uba) Uses ATPAMP+PPi to form thioester bond between its sulfhydryl group and the terminal carboxylate group of UB E2- Ub conjugating enzyme(Ubc):Accepts activated UB E3- Ub Ligase: Catalyzes transfer of UB from E2 to -amino group of lysine on target protein form

16、ing an isopeptide bond 蛋白酶體（蛋白酶體（Proteasome） 19S lid 19S lid 20S core Control of p53 by ubiquitylation vChromatin structure and transcriptional regulation. vTurn over of oncogene products and tumor biology vCell cycle control vRegulation of DNA repair response vApoptosis vEndocytosis 一般了解（不考） Contro

17、l of Cell Cycle Progression by Distinct E3 Complexes Anaphase-Promoting Complex Two families of E3 ubiquitin ligases 一般了解（不考） 一般了解（不考） The Nobel Prize in Chemistry, 2004 “for the discovery of ubiquitin-mediated protein degradation” Avram Hershko Irwin Rose Aaron Ciechanover Questions v細(xì)胞內(nèi)主要的蛋白質(zhì)降解體系是

18、什么？各有 什么特點(diǎn) v人體消化道內(nèi)有哪些蛋白酶參與了蛋白質(zhì)的 消化？ Amino Acid Pool Amino acid metabolism Deamination（脫氨）（脫氨） Transamination（轉(zhuǎn)氨作用）（轉(zhuǎn)氨作用） Urea cycle（尿素循環(huán)）（尿素循環(huán)） Pyruvate group a a Ketoglutarate group Succinyl-CoA group Oxaloacetate group Fumarate / oxaloacetate group Alanine / acetoacetate group Acetyl-CoA / acetoac

19、etate group Decarboxylation 1. Deamination of AAs transamination oxidative deamination union deamination Important in the diagnosis of heart and liver damage caused by heart attack, drug toxicity, or infection. After a heart attack,a variety of enzymes, including these aminotransferases, leak from t

20、he injured heart cells into the bloodstream. Assays for Tissue Damage by Analyses of certain enzyme activities in blood serum轉(zhuǎn)氨酶與器官損傷 Transamination(轉(zhuǎn)氨作用）轉(zhuǎn)氨作用） C R1 H NH3 COO + C R2 O COOC R2 H NH3 COO+C R1 O COO Amino transferases amino acid-1 amino acid-2 keto acid-2keto acid-1 1 An amino group is

21、 transferred to an -keto acid, with formation of the corresponding amino acid and another -keto acid. 2 Usually occurs in amino acid degradation 3 Transamination is reversible, however，transamination of essential amino acid is unidirectional, Ser, Lys and Pro cannot be transaminated. 5 Key roles of

22、Glu and - keto glutarate in transamination Transamination 4 -keto acid produced in transamination can be intermediator for TCA 6 Only the - amino group of Glu enters Urea cycle ALT: Alanine aminotransferase (in liver) 丙氨酸氨基轉(zhuǎn)移酶，丙氨酸氨基轉(zhuǎn)移酶， also called glutamate-pyruvate transaminase（谷丙轉(zhuǎn)氨酶）（谷丙轉(zhuǎn)氨酶）, GPT

23、AST: Aspartate aminotransferase (in heart) ，also called glutamate-oxaloacetate transaminase（谷草轉(zhuǎn)氨酶）谷草轉(zhuǎn)氨酶）, GOT Two important transaminases: 嗜酒如命嗜酒如命 進(jìn)食高蛋白補(bǔ)品進(jìn)食高蛋白補(bǔ)品 服用某些藥物服用某些藥物 肝?。ǜ窝?、肝損傷肝?。ǜ窝?、肝損傷) 腎病腎病( (谷氨酰轉(zhuǎn)肽酶谷氨酰轉(zhuǎn)肽酶) ) /uniprot/P24298 Function Expression Activity Cellular Locali

24、zation Domain Modification Structure Regulation Cofactor Transamination-needs coenzyme pyridoxal phosphate（磷酸吡哆醛，（磷酸吡哆醛， B6） 1 Covalent 2 Interaction 3 Schiffs base Vitamine B6 亞胺 Transamination-need coenzyme pyridoxal phosphate(PLP) ALL Aminotransferases identified so far use pyrodoxal phosphate as

25、 coenzyme. Pyridoxamine phosphate(PMP) (2) Oxidative deamination COOH CHNH2 (CH2)2 COOH L-Glu L-Glu Dehydrogenase NAD+ NADH+H+ COOH C NH (CH2)2 COOH COOH C (CH2)2 COOH H2O NH3 O -ketoglutarate Glutamate Releases Its Amino Group as Ammonia in the Liver 1 In hepatocytes mitochondria, 2 Glutamate under

26、goes oxidative deamination 3 Catalyzed by L-glutamate dehydrogenase 4 Produces free ammonia 5 Produces NADH 6 Required for Urea Cycle 7 The enzyme is strictly regulated How and Why？ (3) Union deamination Alanine + -ketoglutaratePyruvate + glutamate Glutamate + NAD+ + H2O -ketoglutarate + NADH + NH4+

27、 Net Reaction: Alanine + NAD+ + H2O pyruvate + NADH + NH4+ vThe combined action of an aminotransferase and glutamate dehydrogenase vTransaminase, vglutamate dehydrogenase. 草酰乙酸草酰乙酸 腺苷酰琥珀酸腺苷酰琥珀酸 蘋(píng)果酸蘋(píng)果酸延胡索酸延胡索酸 腺苷酸腺苷酸 次黃苷酸次黃苷酸 (3) Purine nucleotide cycle (in muscle) 1. Sources: Deamination of AAs-main

28、 source Catabolism of other nitrogen containing compounds Source and outlet of ammonia （NH3） 2. Outlets: (1) Formation of urea in liver (2) Formation of Gln (3) Synthesis of AA (4) Excrete in urine Metabolism of Ammonia most vertebratesbirds & reptiles fish & other aquatic vertebrates Transportation

29、 of NH3 1 Glucose Alanine cycle 2 Transportation of ammonia by Gln Urea cycle in liver cells Ammonia Is Toxic to Animals 1 Glucose-Alanine Cycle Alanine Transports Ammonia from Skeletal Muscles to the Liver 2. Transportation of ammonia by Gln Excess ammonia in tissues is added to glutamate to form g

30、lutamine, catalyzed by glutamine synthetase(谷氨酰胺合成酶） After transport in the bloodstream, the glutamine enters the liver and NH4 is liberated in mitochondria by the enzyme glutaminase（谷氨氨酰胺酶） blood circulation liver other organs muscle kidney urea glucose alanine cycle Glutaminase Neutral Non-polarit

31、y Non-toxic Easily transported through membrane Formation of urea 1. Site: liver (mitochondria and cytosol) 2. Process - ornithine cycle ornithineNH3 + CO2 H2O NH3H2O H2O urea arginase Arg citrulline Urea Is Produced from Ammonia in Five Enzymatic Steps (氨、瓜、精琥、精、尿） Hans Creb Phospho- carbonate Hydr

32、ogen carbonate Carbamate Carbamoyl- phosphate Formation of Carbamoylphosphate by Carbamoylphosphate Synthetase I Formation of carbamoyl phosphate（氨甲酰（氨甲酰 磷酸）磷酸）in mitochondria +2ADP+Pi 2ATP+CO2+NH3+H2O H2N-C- O 2NH3 + CO2 + 3ATP + 2H2O urea + 2ADP + AMP + 2Pi + PPi 1 The 2 N of urea : NH3 and Asp. 2

33、 Starts/ends up with ornithine 3 NH3, CO2 form caramoyl phosphate, 4 Citrulline formation, 5 Incorperation of Asp. 6 Energe consuming: One urea 3ATP(4 P ?) 8 Needs 5 enzymes： carbamoyl phosphate synthetase ornithine carbamoyl transferase,OCT Argininosuccinate Synthetase Argininosuccinase Arginase 10

34、 Rate limiting :1st step Urea formation: Regulation of urea synthesis Activation of the urea cycle by N-acetyl-glutamate N-acetyl-glutamate synthetase I Regulation of urea synthesis vEnergy consuming: one urea will costs 4 P 1.Carbamoylphasphate synthetase I 2.Substrate activation/induction 3.Respon

35、sive to concentration of ammonia /amino acid in liver 4.Protein-rich dietary 5.Starvation Aspartate（天冬氨酸） Fumarate(延胡素酸） Genetic Defects in the Urea Cycle -Life-Threatening 1.Total lack of any Urea Cycle enzyme is lethal. 2.Hyperammonemia - elevated NH3 in blood. 3.Elevated NH3 is toxic, especially

36、to the brain. 4.Severe mental retardation resulted. Control the protein/amino acid intake Remove the excessive NH3/amino acids Add intermediates of Urea cycle 一般了解（不考） Tetrahydrofolic acid (FH4) One carbon units are carried by FH4. Substance for synthesis of nucleic acid. N5 , N10 of FH4 participate

37、 in the transfer of one carbon units. N H H NN N H2N CH2HN 1 2 3 45 6 7 8 9 10 OH CONH H C COOH CH2CH2COOH 葉酸缺乏： new-born ， Blood cell disease Analog as drug N N 510 CH 2 N5-CH3FH4N5,-CH2-FH4 N10 N N H 5 10 CH3 N N 5 10 CH N5,=CH-FH 4 N10 N H N 510 CHO -CHOFH4N10 N N H 510 CH=NH -CH=NHFH4N5 N H H NN

38、 N H2N CH2HN 1 2 3 45 6 7 8 9 10 OH CONH H C COOH CH2CH2COOH N5 , N10 of FH4 participate in the transfer of one carbon units. Formation of one carbon unit (1) SerN5,N10-CH2-FH4 CH2 CHNH2 COOH + FH4 Ser hydroxymethyl transferase H2O N5, N10-CH2-FH4+ CH2NH2 COOH SerGly (2) GlyN5,N10-CH2-FH4 + FH4 Gly

39、lyase N5, N10-CH2-FH4+ CO2 CH2NH2 COOH + NH3 NAD+ NADH+H+ Gly 一般了解（不考） (3) His N5-CH=NHFH4 NHN CH2CHNH2COOH NH3 NHN CH=CHCOOH 2H2O N HN CH=CHCOOHHOOC-CH FH4 N5-CH=NHFH4 subaminomethyl transferase CHNH2 COOH （CH ） 22 COOH subaminomethyl Glu His Glu 一般了解（不考） (4) TrpN10-CHOFH4 N CH2CHNH2COOH H O2 NHCHO

40、 CCHNH2COOH O N-formyl kynurenine H2O NH2 CCHNH2COOH O HCOOH kynurenine N10 -CHOFH4 synthetase FH2+ATP ADP+Pi N10-CHOFH4 Trp 一般了解（不考） Tyrosine formation from phenylalanine (Monooxygenase reaction, 單氧化酶單氧化酶) PhenylKetonuria苯丙酮尿癥苯丙酮尿癥 1Phenylalanine Hydroxylase 2Tyrosine Aminotransferase 3Parahydroxyp

41、henyle pyruvate Di-oxygenase (ascorbate Vc) 4Homogentisate Dioxygenase 5Maleyl Acetoacetate Isomaerase 6Fumaryl Acetoacetase metabolite concentration in urine mmoles/24 h controlpatient phenylalanine0.181.8 6.0 phenylpyruvate-1.8 12 phenyllactate-1.8 3.3 phenylacetate-sign. incr. phenylacetylglutamate0.8 1.2 9 Phenylketonuria