生物化学教学课件：蛋白质降解及氨基酸分解代谢

1、淮南八公山豆腐制作Department of Biochemistry, University of Colorado(200207-200711)CytokinesisMetaphaseAnaphaseNeuron diversity, dipoid and aneuploidProtein degradation, Amino Acid Metablism：Catablism and Biosynthesis of Amino Acids Nitogen FixationDegradation and Biosynthesis of NucleotidesMetabolism Regula

2、tionLehninger Principles of Biochemistry, 4th editionTextbook of biochemistry with clinical correlations1 Protein digestion2 Protein degradation Lysosome medidated-degradation Autophagy Ubiquitin-mediated3 Amino acid catablism4 Amino acid synthesisProtein degradation, Amino Acid MetablismThe signifi

3、cance of amino acid1. Cells / tissues growing, renewing and mendingProtein Degradation/ Amino Acid MetablismThe significance of amino acid2. Take part in physiological activities3. Oxidation and supply energy70kg的人，每天有400g的protein发生变化：400g发生再循环；100g氧化讲解提供能量4. Nitrogen balanceBefore we get on amino a

4、cid metablism:1. Breakdown of dietary proteins2. Protein degradation in cellsProtein breakdown starts at stomachOptimal pH1.52.5，Low specificityProduce small peptide and free amino acids pepsinogen胃蛋白酶原+ Small PeptideHCl、PepsinPepsinBreakdown of dietary proteins Both are generated in stomach !主细胞（ch

5、ief cell）：胃酶细胞，zymogenic cell壁细胞（parietal cell）：泌酸细胞，oxyntic cell泌酸细胞：较大，多呈圆锥形。核圆而深染，居中；胞质呈均质而明显的嗜酸性。胃酸: 双刃剑，过少或缺乏时，消化不良; 分泌过多,对胃和十二指肠黏膜发生侵袭作用，导致溃疡，穿孔、出血等；Chief cells secrets pepsinogen，典型蛋白质分泌细胞的结构特点：柱状，核圆形；胞质基部呈强嗜碱性，顶部充满酶原颗粒。细胞核周有大量粗面内质网与发达的高尔基复合体。Cholecystokininkolsstkann ( 缩胆囊素) stimulates secre

6、tion of several pancreatic enzymes (胰腺酶）with activity optima at pH 7 - 8.Pancreas exocrine cells :Trypsinogen(胰蛋白酶原）Chymotrypsinogen（糜蛋白酶原）Procarboxypeptidases A, B 羧肽酶The digestion of proteins in the small intestine（小肠）.Enterokinase肠激酶 : produced by cells of the duodenum du:di:nm （十二指肠）丝氨酸蛋白酶（Serin

7、e protease)家族:利用Ser残基来催化水解肽键丝氨酸蛋白酶家族：Trypsin，Chymotrypsin，Elastase，Carboxypeptidases只有一个丝氨酸残基催化肽键断裂，Ser-His-Asp形成催化三联体其它蛋白酶家族：半关氨酸蛋白酶天冬氨酸蛋白酶金属离子蛋白酶丝氨酸蛋白酶1 丝氨酸蛋白激酶通过有限水解，调控（激活）丝氨酸蛋白水解酶；2 丝氨酸蛋白水解酶有相似的结构和功能；在肽键水解过程中表现高特异性；3 调控血液凝固，补提激活，受精，激素生产等许多生理过程；4 动物/植物中存在天然抑制剂；5 调控缺乏或异常导致蛋白质消化自溶，从而发生疾病，如血友病、癌症转移等

8、。Synthesis of the enzymes as inactive precursors-zymogen(酶原或前酶）:1 Protects the exocrine cells from destructive proteolytic attack. 2 Transportation3 Allow RegulationElaborate mechanism for getting active digestive enzymes.Protein Digestion Proteolytic enzymes of pancreatic juiceEndopeptidasesExopept

9、idasesTrypsin : Arg, Lys (C)Chymotrypsin : Tyr, Trp, Phe, Met, Leu (C)Elastase: Ala, Gly, Ser (C)carboxypeptidaseaminopeptidaseCleavage sites and substrates of proteasesMARTKQTARKSTGGKAPRKQLATKAARKSAPSTGCleavage sites and substrates of proteases一They hydrolyze the peptides very efficiently.In humans

10、, most globular proteins from animal sources are almost completely hydrolyzed to amino acids in the gastrointestinal tract, some proteins, such as keratin(角蛋白）are only partly digested. The protein content of some plant foods is protected against breakdown by indigestible cellulose husks（外壳）.Absorpti

11、on of free amino acids Trypsin and Protease in Cell CultureTrypsin used for protein sequencing Enterokinase used in protein purification 肠激酶酶切位点DDDDK 基于MS/MS的蛋白质序列和修饰鉴定Liquid chromatography-coupled mass spectrometery(LC-MS)Tryspin digestionA disease caused by obstruction of the normal pathway by whi

12、ch pancreatic secretions enter the intestine. The enzymogens of the proteolytic enzymes are converted to their catalytically active forms prematurely, inside the pancreatic cells, and attack the pancreatic tissue itself. This causes excruciating kskru:etl pain and damage to the organ that can prove

13、fatal. Acute pancreatitis and Protease(急性胰腺炎）Questions：1 蛋白酶水解的是什么化学键？2 人体消化道内有哪些蛋白酶? 3 酶以酶原的形式存在有什么意义？Protein Degradation in CellLife-span of protein in cell：Insulin(胰岛素)：1hrhemoglobin、Histone：few monthsLens, neuron：lifelongMost of proteins: a few daysProtein degradation in cells:1 dynamic turnover

14、 :2 apoptosis:Protein degradation in cells:1 dynamic turnover :2 apoptosis:3 Energy-providing:机体中大多数的蛋白和氨基酸存在与骨骼肌中，机体运耗能时，这些蛋白质被降解为氨基酸，用于产能和糖异生。4 Branch chain amino acids are preferred for energy-providing.5 Protein degradation is occurring everywhere in the body, muscle is the major location of pro

15、tein degradation.Two major systems for the turnover of cellular proteins:Lysosomes and Ubiquitin-mediated protein degradationProtein Degradation in Cell1 Lysosomal hydrolasesexhibit acidic pH optimal.2 Lost activity if leaked into cytosol.3 Cathepsin 组织蛋白酶家族Low specificityLysosomal Degradation Pathw

16、ayProteinsA. EndocytosedB. Cytosolic C. Organelles by autophagy溶酶体贮积病：基因突变致溶酶体酸性水解酶缺陷痛风：溶酶体破裂大隅良典Yoshinori Ohsumi，2016年诺贝尔生物学和医学奖, Prize motivation: for his discoveries of mechanisms for autophagyAutophageConsequences of autophagy：survival or cell deathConsequences of autophagy：survival or cell deat

17、h自噬作用是细胞内的溶酶体降解自身细胞器和其他大分子的过程。形态学特征：电镜下可观察到胞质中有大量包裹着细胞浆和细胞器的空泡结构，称作自噬泡。主要过程：自噬泡形成，与溶酶体发生融合，自噬泡所包裹着的待降解物质进入溶酶体，在各种酶的作用下分解成氨基酸和核苷酸等，并进入三羧酸循环，产生小分子和能量ATP-再被细胞所利用Autophagic cell death and apoptosis细胞自噬与细胞凋亡之间似乎存在着某种调控机制，可调节二者的相互拮抗或促进相互替换Autophagy and drug resistence(细胞耐药性)细胞自噬对人类健康是一把双刃剑。Ubiquitin pathw

18、ayUbiquitinProteasomeE3 ligase(Cullin-RING complex)Protein Degradation System in CellWhat is Ubiquitin and Ubiquitylation?Human Ub: MQIFKVTLTGKTITLEVEPNDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGGYeast Ub: MQIFKVTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG蛋白酶体（P

19、roteasome）19S lid19S lid20S coreUbiquitination Ub activating enzyme(Uba) Ub conjugating enzyme(Ubc) E3- Ub Ligase-SubUbiquitin pathwayE1-S-UbE2-S-UbE3substratesUbMono-ubiquitinationChange：protein conformation,Localization, interactionPolyubiquitinationUbUbUbUbsubstratesProteosomeDegradationE1- Ub ac

20、tivating enzyme(Uba)Uses ATPAMP+PPi to form thioester bond between its sulfhydryl group and the terminal carboxylate group of UBE2- Ub conjugating enzyme(Ubc):Accepts activated UBE3- Ub Ligase: Catalyzes transfer of UB from E2 to -amino group of lysine on target protein forming an isopeptide bond异肽键

21、ATP肽键和异肽键substrateUbMQIFKVTLTGKTITLEVEPNDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG蛋白质泛素化降解途径7要素Control of p53 by ubiquitylationChromatin structure Transcriptional regulation.Turn over of oncogene productsCell cycle controlRegulation of DNA repair responseApoptosisEndocytosis一般了解（不考）Con

22、trol of Cell Cycle Progression by Distinct E3 ComplexesAnaphase-Promoting ComplexTwo 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 HershkoIrwin RoseAaron CiechanoverQuestions细胞内主要的蛋白质降解体系是什么？细胞内主要的

23、蛋白质降解体系各有什么特点?泛素化降解的要素有哪些？消化道内有哪些蛋白酶参与了蛋白质的消化？Amino Acid PoolAmino acid metabolism Deamination（脱氨） Transamination（转氨作用） Urea cycle（尿素循环） Pyruvate group a Ketoglutarate group Succinyl-CoA group Oxaloacetate group Fumarate / oxaloacetate group Alanine / acetoacetate group Acetyl-CoA / acetoacetate gro

24、up Decarboxylation1. Deamination of AAstransamination oxidative deaminationunion deamination Important in the diagnosis of heart and liver damage caused by heart attack, drug toxicity, or infection. After a tissue/organ damage, a variety of enzymes, including these aminotransferases, leak from the i

25、njured tissue cells into the bloodstream. Assays for Tissue Damage by Analyses of amino-transferase activities in blood serumTransamination(转氨作用）1 An amino group is transferred to an -keto acid, with formation of the corresponding amino acid and another -keto acid.2 Usually occurs in amino acid degr

26、adation3 Transamination is reversible, however，transamination of essential amino acid is unidirectional, Ser, Lys and Pro cannot be transaminated.5 Key roles of Glu and -keto glutarate in transaminationTransamination4 -keto acid produced in transamination can be intermediator for TCA 6 Only the - am

27、ino group of Glu enters Urea cycleALT: Alanine aminotransferase (in liver) 丙氨酸氨基转移酶，also called glutamate-pyruvate transaminase（谷丙转氨酶）, GPTAST: Aspartate aminotransferase (in heart) ，also called glutamate-oxaloacetate transaminase（谷草转氨酶）, GOTTwo important transaminases:嗜酒如命进食高蛋白补品服用某些药物肝病（肝炎、肝损伤)肾病(

28、谷氨酰转肽酶)/uniprot/P24298Function ExpressionActivityCellular Localization DomainModificationStructureRegulationCofactorTransamination-needs coenzyme pyridoxal phosphate（磷酸吡哆醛， B6）1 Covalent ligated to enzyme2 Interaction with enzyme3 Schiffs baseVitamine B6亚胺Transamination need coenzyme pyridoxal phosp

29、hate(PLP)ALL Aminotransferases identified so far use pyrodoxal phosphate as coenzyme. Pyridoxamine phosphate(PMP)(2) Oxidative deaminationGlutamate Releases Its Amino Group as Ammonia in the Liver1 In hepatocytes mitochondria, 2 Glutamate undergoes oxidative deamination 3 Catalyzed by L-glutamate de

30、hydrogenase4 Produces free ammonia5 Produces NADH 6 Required for Urea Cycle7 The enzyme is strictly regulated How and Why？(3) Union deaminationAlanine + -ketoglutaratePyruvate + glutamateGlutamate + NAD+ + H2O-ketoglutarate + NADH + NH4+Net Reaction: Alanine + NAD+ + H2Opyruvate + NADH + NH4+The com

31、bined action of an aminotransferase and glutamate dehydrogenaseTransaminase, glutamate dehydrogenase.(3) Purine nucleotide cycle (in muscle)草酰乙酸腺苷酰琥珀酸苹果酸延胡索酸腺苷酸次黄苷酸1. Sources: Deamination of AAs-main source Catabolism of other nitrogen containing compoundsAmmonia (NH3) :Source and outlet2. Outlets:(

32、1) Formation of urea in liver(2) Formation of Gln(3) Synthesis of AA(4) Excrete in urineMetabolism of Ammoniamost vertebratesbirds & reptilesfish & other aquatic vertebratesTransportation of NH31 Glucose Alanine cycle2 Transportation of ammonia by Gln Ammonia Is Toxic to Animals!Ammonia is converted

33、 to Urea cycle in liver cells.Life-span of protein in cell：Insulin 胰岛素：1hrhemoglobin、Histone：few monthsLens, neuron：lifelongMost of proteins: a few daysProtein degradation in cells:1 dynamic turnover :2 apoptosis:3 Energy-providing:机体中大多数的蛋白和氨基酸存在与骨骼肌中，机体运耗能时，这些蛋白质被降解为氨基酸，用于产能和糖异生。4 Branch chain ami

34、no acids are preferred for energy-providing.5 Protein degradation is occurring everywhere in the body, muscle is the major location of protein degradation.1 Glucose-Alanine CycleAlanine Transports Ammonia fromSkeletal Muscles to the Liver2. Transportation of ammonia by GlnExcess ammonia in tissues i

35、s added to glutamate to form glutamine, 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 circulationliver otherorgans musclekidneyureaglucose alanine cycleGlutamin

36、ase丙氨酸：NeutralNon-polarityNon-toxicEasily transported through membraneGlutamine synthetaseFormation of urea1. Site: liver (mitochondria and cytosol)2. Process - ornithine cycleUrea Is Produced from Ammonia in Five Enzymatic Steps(氨、瓜、精琥、精、尿）Hans CrebPhospho-carbonateHydrogencarbonateCarbamateCarbamo

37、yl-phosphateFormation of Carbamoylphosphateby Carbamoylphosphate Synthetase I Formation of carbamoyl phosphate（氨甲酰磷酸）in mitochondria+2ADP+Pi2ATP+CO2+NH3+H2OH2N-C-PO1 The 2 N of urea : NH3 and Asp.2 Starts/ends up with: ornithine3 NH3, CO2 form caramoyl phosphate,4 Citrulline formation,5 Incorporatio

38、n of Asp.6 Energy consuming: One urea 3ATP, 4 P7 Needs 5 enzymes：carbamoyl phosphate synthetase ornithine carbamoyl transferase, OCTArgininosuccinate SynthetaseArgininosuccinaseArginase8 Rate limiting :1st step9 mitochondrianl vs cytosolUrea formation:Regulation of urea synthesisActivation of the ur

39、ea cycle by N-acetyl-glutamateN-acetyl-glutamate synthetase IRegulation of urea synthesisEnergy consuming: one urea will costs 4 PCarbamoylphasphate synthetase ISubstrate activation/inductionResponsive to concentration of ammonia /amino acid in liverProtein-rich dietary Starvation Aspartate（天冬氨酸） Fu

40、marate(延胡素酸）Genetic Defects in the Urea Cycle -Life-ThreateningTotal lack of any Urea Cycle enzyme is lethal.Hyperammonemia - elevated NH3 in blood. Elevated NH3 is toxic, especially to the brain. Severe mental retardation resulted.Control the protein/amino acid intakeRemove the excessive NH3/amino

41、acidsAdd intermediates of Urea cycle一般了解（不考）Tetrahydrofolic acid (FH4) and one carbon unitOne carbon units are produced in catablite of aa and carried by FH4. One carbon units are substance for synthesis of nucleic acid.In great need of new-born蝶啶对氨基苯甲酸GlutamatePurine ring N5 , N10 of FH4 participat

42、e in the transfer of one carbon units. Analog of FH4 as drug for anti-bacterial蝶啶对氨基苯甲酸谷氨酸Formation of one carbon unit(1) SerN5,N10-CH2-FH4 (2) GlyN5,N10-CH2-FH4甘氨酸剪切一般了解（不考） (3) His N5-CH=NHFH4一般了解（不考）(4) TrpN10-CHOFH4一般了解（不考）支链氨基酸的起始途径是相同的：BCAA aminotransferases 同工酶Branched keto acidsDehydrogenase

43、1 BCAA aminotransferases are richer in muscle；2 Fasting induces the activity of these enzymes in muscle, but not those in liver;3 BCAA aminotransferases with higher activity locate in liver after dinner.支链氨基酸分解代谢酶缺乏并不多见，一旦发生可造成酸中毒；支链酮酸脱氢酶复合体活性缺失导致枫树蜜汁尿病：尿液具有特殊的枫树蜜汁气味，伴随酸中毒，智力迟缓和短寿。限制膳食治疗！Tyrosine fo

44、rmationfrom phenylalanine(Monooxygenase reaction,单氧化酶)PhenylKetonuria苯丙酮尿症Phenylalanine HydroxylaseTyrosine AminotransferaseParahydroxyphenyle pyruvate Di-oxygenase (ascorbate Vc)Homogentisate DioxygenaseMaleyl Acetoacetate IsomaeraseFumaryl Acetoacetasemetaboliteconcentration in urine mmoles/24 hco

45、ntrolpatientphenylalanine0.181.8 6.0phenylpyruvate-1.8 12phenyllactate-1.8 3.3phenylacetate-sign. incr.phenylacetylglutamate0.8 1.2 9Phenylketonuria(苯丙酮尿症， PKU ）: elevated metabolites in urineNon-functional mutation of the phenylalanine hydroxylase gene. There is no cure for PKU If the condition is

46、left untreated, it can cause problems with brain development, leading to progressive mental retardation, brain damage, and seizures. A low-phenylalanine diet. Maintain blood Phe levelsPhenylketonuria (PKU). Tyrosine metablism derivates, parkinson, albinismmelanin褪黑激素血清素脑中的神经递质，动脉和支气管平滑肌收缩诱导睡眠，晚间合成松果

47、体，视网膜一般了解（不考）How about a cup of milk before sleep?百忧解，氟西汀，Fluoxetine，其药物形态为盐酸氟西汀（Fluoxetine hydrochloride），抗忧郁药，商品名为“百优解”或“百忧解”（Prozac）。是一种选择性5-羟色胺再吸收抑制剂（SSRI），在临床上用于成人忧郁症、强迫症，广场恐惧症和神经性贪食症的治疗。一般了解（不考）acceptorMethionine metabolismI一般了解（不考）高半胱氨酸甲基受体接受甲基甲硫氨酸腺苷甲硫氨酸Methylatedacceptor高半胱氨酸Cysteine is not

48、an essential amino acid if methionine is availableMethionine metabolismII一般了解（不考）Catabolites of amino acid -KetoglutarateSuccinyl CoA（琥珀）Fumarate延胡索酸OxaloacetatePyruvateAcetyl CoAAcetoacetyl CoAIntermediates of TACPEPGlucoseFatty acidKetone bodies blood circulationliver otherorgans musclekidneyureaQ

49、uestions:1 氨基酸分解进入三羧酸循环之前要先经过什么过程？2 氨基酸脱氨有哪几种类型？转氨酶的辅基是什么?3 氨基转运有哪二种类型，什么是葡萄糖丙氨酸循环？4 氨基酸脱下的氨在人体体内的主要去路有哪些，最终产物是什么？5 尿素循环的大概过程，消耗的能量，尿素中N的来源是怎样的?6 一碳单位的载体是什么？ Biosynthesis of Amino Acid and Physiologic Amines？AlanineAsparagineAspartateCysteineGlutamateGlutamineGlycineProlineSerineTyrosineArginine*His

50、tidineIsoleucineLeucineLysineMethioninePhenylalanineThreonineTryptophanValineEssential and Nonessential Amino Acids for Humans Essential Nonessential*Arg is essential in infants and growing children but not in adults. Most bacteria and plants can synthesize all 20 amino acids.AAs that an organism ca

51、nnot synthesize under a given set of conditions are called “essential.” Those that can be synthesized in adequate amounts are “nonessential.”23 Humans can synthesize 10 of the 20 common amino acidsBiosynthetic pathways for AAs:Six families corresponding to their metabolic precursorsa-Ketoglutarate G

52、ives Rise to Glutamate, Glutamine,Proline, Arginine氨浓度很高时Oxidative deaminationGlutamate Releases Its Amino Group as Ammonia in the Liver1 In hepatocytes mitochondria, 2 Glutamate undergoes oxidative deamination 3 Catalyzed by L-glutamate dehydrogenase4 Produces free ammonia5 Produces NADH 6 Required

53、 for Urea Cycle7 The enzyme is strictly regulatedGlutamate and GlutamineGlutaminaseGlutamine synthetaseProline is a cyclized derivative of glutamate吡咯碄-5-羧酸还原酶吡咯-5-羧酸合成酶合成酶缺乏：Too much pro. in blood白内障智力迟钝皮肤过弹性脱氢酶缺乏：Too much pro，良性，但是会诱发癫痫脱氢酶还原酶Urea cycleAAGluGluCarbamoylphosphateornithinecitrullinec

54、itrullineArgininosuccinateornithinArginineOxaloacetateAAGluAspartate2ADP+Pi2ATP+CO2+NH3+H2O1cytosolmitochondrionNH2-C-NH2OUreaa-ketoglutarateH2N-C-PO2345a-ketoglutaratea-ketoglutarateArginineIn liverArginine synthesis in kidney（no arginase)Serine, Glycine, Are Derived from 3-PhosphoglycerateCysteine

55、 Are Derived from 3-Phosphoglycerate （through serine）Mammals synthesize cysteine from homocysteine(高半胱氨酸） and serine.Homocysteine，Methionine, S-adenosylmethionine,S-adenosylhomocysteine腺苷甲硫氨酸腺苷高半胱氨酸甲硫氨酸高半胱氨酸Three Nonessential and Six Essential Amino Acids Are Synthesized from Oxaloacetate and Pyruva

56、teEssential AAs: Methionine, threonine, lysine, isoleucine, valine, leucine Nonessential AAs:AlanineAspartateAsparagineAsparagine is synthesized by amidation of aspartate, with glutamine donating the NH4 .Tyrosine formationfrom phenylalanine(Monooxygenase reaction)Tyrosine is considered a conditiona

57、lly essential amino acid, or as nonessentialLysineMethionine一般了解（不考）IsoleusineLeusineValine一般了解（不考）Main biological route of aromatic ring formation. Ring closure of an aliphatic precursor followed by stepwise addition of double bonds.Synthesis of Tryptophan, Phenylalanine, Tyrosine一般了解（不考）Chorismate

58、 （分支酸）Is a Key Intermediate in the synthesis of Tryptophan, Phenylalanine, and Tyrosine1 An aliphatic precursor :Shikimate2 Stepwise addition of double bonds3 Addition of three more carbons. 一般了解（不考）Chorismate Is a Key Intermediate in the synthesis of Tryptophan, Phenylalanine, and Tyrosine一般了解（不考）C

59、horismate and Prephenate to Phenylalanine, Tyrosine一般了解（不考）Histidine Biosynthesis Uses Precursors of Purine BiosynthesisThe pathway to histidine in all plants and bacteria differs in several respects from other amino acid biosynthetic pathways. Histidine is derived from three precursors: PRPP contri

60、butes five carbons, thepurine ring of ATP contributes a nitrogen and a carbonand glutamine supplies the second ring nitrogen. 一般了解（不考）N-1 of the purine ring is linked to the activated C-1 of the ribose of PRPP; Condensation of ATP and PRPPpurine ring opening that ultimately leaves N-1 and C-2 of ade