生物化學：Chapter 18 Amino Acid Oxidation

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1、Chapter 18 Amino Acid Oxidation and the Production of Urea1. Nitrogen excretion and the urea cycle2. Pathways of amino acid degradation Lectured by Dr. Qin Yongmei (秦詠梅): Dec. 4 & 6, 2012 Overview of amino acid catabolismH - C - COO-RNH3Amino groupCarboxyl group Removal of the amino groups by transa

2、mination The left carbon skeletons are converted to common intermediates of energy-producing pathways. Catabolism of amino acids usually begins by removing the amino groups, which are then be excretedOverview of amino acid catabolismu Dietary proteins - primary source of amino acids (AAs) uAAs from

3、digestion of dietary proteins are absorbed through intestinal epithelium into the blood;u Various cells take up AAs, which enter cellular pools;u AAs are used as synthesis of cellular proteins and other nitrogen-containing compounds;u Before AA oxidation can occur, the amino nitrogen must be removed

4、;u AA nitrogen forms ammonia, which is toxic to body;uAAs are converted to urea for excretion (the urea cycle)uExcess AAs cannot be stored ! Surplus AAs are used as fuel. Most of carbons are converted to pyruvate, intermediates of TCA cycle, or to acetyl CoA; During fasting state (in the liver), the

5、 carbons are converted to glucose (glucogenic amino acids) or ketone bodies (ketogenic amino acids) and released into the blood for oxidation elsewhere, ultimately, the carbons are all converted into CO2 and H2O via the TCA cycle.uAAs may also be converted to FAs and stored as TGs in adipose tissue;

6、u The liver is the major site for AA oxidation (but most tissues can oxidize branched chain amino acids, e.g. leu, ile, val).Protein digestion and amino acid adsorption Proteolytic enzymes (proteases) break down dietary proteins into their constituent AAs (in the stomach and intestine); Many proteas

7、es are synthesized as inactive zymogens, which are secreted and cleaved to their active protease form; In the stomach, pepsin begins the process by breaking down proteins to smaller polypeptides Enzymes (e.g. trypsin 胰蛋白酶胰蛋白酶, chymotrypsin 胰凝乳蛋白酶胰凝乳蛋白酶, elastase 彈性酶彈性酶, ,carboxypeptidases 羧肽酶羧肽酶) pr

8、oduced by the exocrine pancreas act in the small intestine to continue the process, generating oligopeptides and AAs. AA absorption occurs through the intestinal epithelium; AAs are transported across the plasma membrane into blood. Amino group catabolism(ammonia is processed for excretion in liver)

9、aminotransferasesGlutamate dehydrogenaseUrea and Uric acidu About 90% of nitrogen of yours is normally excreted as urea.u Over a 24 hour period, you would excrete about 9 g of urea.u You also have uric acid (250-750 mg): the degradation product of purine bases; (adenine, guanine, hypoxanthine, xanth

10、ine) High concentration of uric acid leads to gout (痛風)Enzyme-catalyzed transaminations(this reaction is fully reversible !)轉(zhuǎn)氨酶轉(zhuǎn)氨酶 - -酮戊二酸酮戊二酸L-L-谷氨酸谷氨酸Glutamate-ketoglutarateSchiff base=Glu is oxidatively deaminated (catalyzed by glutamate dehydrogenase谷氨酸脫氫酶 in liver mitochondria) to yield ammoniu

11、m ion (NH4+) and regenerate -ketoglutarateOxidative deamination氧化脫氨基氧化脫氨基How do you understand the function of glutamate dehydrogenase ?機體借助聯(lián)合脫氨基作用迅速脫氨基機體借助聯(lián)合脫氨基作用迅速脫氨基Free ammonia (which is toxic) produced in extrahepatic tissues is added to Glu to form Gln which is then transported to liver.(Gln a

12、lso serves as a source of amine groups in biosynthesis)Extrahepatic tissueLiver mitochondriaGlucose-alanine cycleAlanine brings both carbonand nitrogen from muscleto liver PLP acts as a temporary carrier of amino groups at the active sites of all aminotransferases (covalently bound to the enzyme act

13、ive site through an aldimine linkage to the amino groupof a Lys residue). PLP undergoes reversible transformation between its aldehyde form and its aminated form. PLP facilitates several different types of transformation aroundthe -carbon of amino acids.PLP: coenzyme of aminotransferase (PMP)Asparta

14、te aminotransferase (dimer)PLP is covalently linked to a specific Lys residue in the active sites of aminotransferases(Aldimine linkage)Active site of aspartate aminotransferase 2-methyl-Asp-PLP(substrate analog)LysPLPReactions at C:1、racemization2、decarboxylation3、transaminationH - C - COO-RNH3Mult

15、iple roles of PLPReactions at C: tryptophan synthetase;Reactions at C: cytathionine -synthasePLP mediates aminotransferase reactiontransaminationPLP acts as an e- sinkdecarboxylationracemizationTransamination occurs via a Ping Pong mechanism Ping Pong mechanism: the first substrate reacts and the pr

16、oduct must leave the active site before the second substrate can bind. The protonated form of PLP acts as an electron sink to stabilize catalytic intermediates that are negatively charged. The incoming AA binds (I substrate) to the active site, donates its amino group to PLP, and departs in the form

17、 of -keto acid (I product); The incoming -keto acid (II substrate) then binds, accepts the amino group from PMP, and departs in the form of an amino acid (II product).Assays for tissue damage (example: aminotransferases )Damaged heart or liver cells leak aminotransferases into bloodstream. Aspartate

18、 aminotransferase (AST; glutamate-oxaloacetate transaminase, GOT) Alanine aminotransferase (ALT; glutamate- pyruvate transaminase, GPT)Sources From amino acids: aminotransferases and glutamate dehydrogenase; From glutamine: glutminase (kidney, intestine) From bacterial action in the intestine: ureas

19、e From amines: amine oxidase From purines and pyrimidines: amino groups attached to the rings of purines and pyrimidines Ammonia is toxic to animalsTransport of ammonia1. Urea: liver kidney excretion2. Glutamine: muscle, liver and CNS Glutamine is removed by the kidney and deaminated by glutaminase3

20、. Alanine: Glucose-alanine cycle Excess amount of ammonia in the blood (1000 M/L)Causes: liver function problems (urea cylce, liver diseases)Syndrome: tremors, slurring of speech, somnolence (嗜睡), vomiting, cerebral edema (腦水腫), blurring of vision Hyperammonemia (高氨血癥)Possible mechanism Glutamate de

21、hydrogenase reaction is shifted toward synthesis glutamate, resulting in the depletion of TCA intermediates and deprivation of brain energy. Glutamine is the main source of glutamate (neurotransmitter precursor) and is produced by glial cells (神經(jīng)膠質(zhì)細胞). Ammonia inhibits the activity of glutaminase, r

22、esulting in the neurotransmitter (e.g. glutamate, -aminobutyrate) deficiency.Discovery of the Urea Cycle (Krebs and Hensenleit, 1932)The first hypothesis represented in a two-step reactions:Amino Acid + X NH3Ammonia + YNH3 + Y Urea + Z The potential precursors tested: alanine, phenylalanine, glycine

23、, thymine, thymosine, uridine, uracile, cystein, arginine, methylamine, cystine, ammonia carbamine, ammonia cyanate, asparagine, aspartate Addition of arginine yielded the substantial rate of urea formation; But, the result was not exciting, because: The low frequency of arginine in proteins could n

24、ot account for the high amount of urea production; The arginase reaction also occurred in tissues except liver. A modified working hypothesis:Amino acid + NH3 Urea + Z Slices of rat liver can bring about the net conversion of ammonia to urea and the synthesis is markedly stimulated when ornithine is

25、 added. The amount of urea synthesized greatly exceeds the amount of ornithine added (i.e., ornithine acts catalytically). ornithine2NH3 + Ornithine + X YY Urea + Z Working hypothesis was modified again:How were ornithine and ammonia converted to arginine ?？via citrulline NH4+ in hepatocytes is conv

26、erted into urea for excretion via the urea cycle (first metabolic cycle discovered in 1932) in most terrestrial vertebratesThe Urea Cycle ( occurs in liver !)Sourses of NH4+The synthesis ofcarbamoyl phosphate requires two activationsteps, consuming twoATP molecules: onefor activating HCO3-,the other

27、 to phosphorylate carbamate.The reaction catalyzed by carbamoyl phosphate synthetase I瓜氨酸瓜氨酸鳥氨酸鳥氨酸精氨琥珀酸精氨琥珀酸延胡索酸延胡索酸Ornithinetranscarbamoylase(鳥氨酸氨甲酰轉(zhuǎn)移酶)Argininosuccinate synthetaseArgininosuccinatelyase 精氨酸精氨酸The reaction catalyzed by argininosuccinate synthetase(精氨琥珀酸合成酶精氨琥珀酸合成酶)second nitrogenent

28、ers from aspartateUREAGropper et al. 2005 Overall reaction of urea cycle: CO2 + NH4+ + 3ATP + Asp + 2H2O urea + 2ADP + 2Pi + AMP + PPi + fumarate Urea consists of one nitrogen atom from aspartate, one nitrogen atom from NH4+, and one carbon from CO2. Energy expensive disposal, 2ATP per NH3 or 4ATP p

29、er urea (AMP equivalent to 2ATP) But, regeneration of oxaloacetate produces NADH (2.5ATP) Insufficiency of the urea cycle occurs in some genetic disorders and in liver failure.ureaThe puzzle of Urea Cycle: Why the individual urea cycle enzymes were found outside of the liver ?Interorgan relationship

30、s inurea and arginine synthesisIUBMB Life 52: 265-270Endogenous synthesis of arginine or citrulline43Synthesis of Nitric Oxide (NO)NH3+NH2H2N=C-HNCH2CH2CH2CHCO2-+NH3+NH2CONHCH2CH2CH2CHCO2-Nitric oxide synthase (NOS)NH3+NH2H2N=C-HNCH2CH2CH2CHCO2-+ArginineNH3+NH2CONHCH2CH2CH2CHCO2-Citrulline+ NO + NAD

31、P+ + H2ONitric oxide, known as the endothelium-derived relaxing factor ( (內(nèi)皮源性舒張因子內(nèi)皮源性舒張因子) )+ NADPH, O2ASS: argininosuccinate synthetase; ASL: argininosuccinate lyase ; ADC, arginine decarboxylase; AGAT, arginine:glycine amidinotransferase; ARG, arginase; OAT, ornithine aminotransferase; ODC, ornit

32、hine decarboxylase; OTC, ornithine transcarbamylase; P5C, L- 1-pyrroline-5-carboxylate Metabolic sources and fates of arginine, citrulline, and ornithine J. Nutr. 134:2743S-2747S Urea Cycle link to TCA After aspartate enters urea cycle and is deaminated, fumarate is produced. Fumarate enters TCA oxa

33、loacetate (fumarate links the two cycles !) Oxaloacetate can have several fates: condensation with acetyl CoA citrate Oxaloacetate PEP gluconeogenesis pyruvate . Transamination to aspartate back to urea cycleLinks between the urea cycle and citric acid cycleFumarate is converted back to Asp via a pa

34、rtial usage of the citric acid cycle.Allosteric regulation:N- acetyl glutamate positively regulates carbamoyl phosphate synthetase I activity: - synthesized as glutamate builds up - urea cycle accelerated with fast AAs break down Gene regulation: Syntheses of the urea cycle enzymes are all increased

35、 during starvation (when energy has to be obtained from muscle proteins !) or after high protein uptake. The rates of transcription of the five genes encoding the enzymes are increased.Urea Cycle - regulation Carbamoyl phosphate synthetase I is positively regulated by the enigmatic N-acetyl-glutamat

36、eArginineTreatment of deficiencies in urea cycle enzymes by administration of benzoate and phenylbutyrate(excreted in urine)(excreted in urine)What does mean when high levels of urea in the blood occurs ? 1. In general, urea is produced and excreted at a roughly constant rate.2.Over-production of ur

37、ea in the body rarely happend.3.The most common cause of uremia 尿毒癥 is renal problems (chronic renal failure, 慢性腎衰竭 or hypothyroidism 甲狀腺機能減退 ). 4. Urea levels can also be increased in some malignant blood disorders, (e.g. leukaemia 白血病 and multiple myeloma 多發(fā)性骨髓瘤). Pathways of amino acid degradatio

38、nHistory of Dietary Protein-Derived Glucose Production Experiment: The rabbits or dogs were treated by renal glucose transport inhibitor (phlorphizin); measure D/N ratio: glucose (dextrose) to nitrogen ratio the amount of glucose in the urine as a indication of amount of protein digested. D/N=3.75:

39、3.75 g glucose/1 g N IUBMB Life (2010) : 62: 660-668Peripheral glucose concentration did not increaseafter protein ingestion in normal cases Ingestion of 50-100g protein to people (with or without type 2 diabetes) caused an crease in insulin concentration; Administration of individual or mixtures of

40、 amino acids, stimulated a rise in glucagon concentration or a rise in insulin concentration. IUBMB Life (2010) : 62: 660-668Excess amino acids are not degraded by 20 different pathways. The catabolism of all the amino acids converges to seven common metabolic intermediates that feed into the TCA cy

41、cles: pyruvate-ketoglutaratesuccinyl-CoAfumarateoxaloacetateacetyl-CoAacetoacetateGlucogenic amino acids: Their carbon skeletons are degraded to pyruvate, or to one of the 4- or 5-carbon intermediates of TCA Cycle that are precursors for gluconeogenesis. Glucogenic amino acids are the major carbon s

42、ource for gluconeogenesis when glucose levels are low. They can also be catabolized for energy or converted to glycogen or fatty acids for energy storage. Ketogenic amino acids: Their carbon skeletons are degraded to acetyl-CoA or acetoacetate. Acetyl CoA, and its precursor acetoacetate, cannot yiel

43、d net production of oxaloacetate, the precursor for the gluconeogenesis pathway. For every 2-C acetyl residue entering TCA Cycle, two carbon atoms leave as CO2. Carbon skeletons of ketogenic amino acids can be catabolized for energy in TCA Cycle, or converted to ketone bodies or fatty acids. They ca

44、nnot be converted to glucose. Some amino acids are both glucogenicand ketogenic:IlePheTyrTrpBoth glucogenic & ketogenic Leucine and lysine are purely ketogenic AAs All others are glucogenic AAs.One-carbon unit carriers act in amino acid metabolismThree types of one-carbon unitcarriersCarrier of the

45、most oxidizedCarrier of the most oxidizedone carbon unitone carbon unitCarrier of the mostCarrier of the mostreduced one carbon unitreduced one carbon unit one carbon unit of one carbon unit of intermediate oxidation statusintermediate oxidation status-CH4-CO2Conversion of one-carbon units ontetrahy

46、drofolateSer, GlyHis甲基甲基羥甲基羥甲基甲?；柞；鵅ecause of the essential roles of THF as acceptor and donor of single carbon units, dietary deficiency of folate, genetic deficiencies in folate metabolism or transport, and the increased catabolism of folate seen in some disease states, result in various metaboli

47、c effects leading to increased risk of developmental defects, cardiovascular disease, and cancer. Synthesis of methionine and S-adenosylmethionine in an activated-methyl cycleExamples of the role of S-adenosylmethionine (SAM) as methyl group donor:Methylation of bases in tRNA ;Methylation of cytosin

48、e residues in DNA ;Methylation of norepinephrine to form epinephrine ;Conversion of the glycerophospholipid phosphatidylethanolamine to phosphatidylcholine . a、to acetyl-CoA via pyruvate Alalnine, Cysteine, Glycine, Serine, Threonine, Tryptophanb、to acetyl-CoA via acetoacetyl-CoA Leucine, Lysine, Ph

49、enylalanine, Tryptophan, Tyrosine 1. The amino acids are degraded to acetyl-CoA6 AAs are degraded to pyruvate. Serine dehydratase reaction 烯胺烯胺亞胺亞胺Serine Hydroxymethyltransferase Reaction predominate in animalsGlycine Cleavage EnzymeReaction predominate in animalsCrystals of calcium oxalate account

50、for up to 75% of all kidney stones(kidney)Alanine: to pyruvate directly by transamination;Tryptophan: cleaved to alanine as one part;Threonine: cleaved to acetyl-CoA + glycine (10-30% of threonine catabolism, major pathway in humans lead to succinyl-CoA)Glycine: (1) to serine; (2) to CO2 + NH4+; (3)

51、 to glyoxylate oxylateSerine: serine dehydratase (remove -amino group and -hydroxy group in a single PLP-dependent reaction )Cysteine: via two steps (remove sulfur atom and transamination) 7 AAs are degraded to acetyl-CoAC pyruvate or TCA intermediatesC acetyl-CoATryptophanLysine-KetoadipateAcetoace

52、tyl-CoAPhenylalanineTyrosineLeucineFumarateAcetoacetateAcetyl-CoA-Methylgluta-conyl CoAAlaninepyruvateIntermediates of tryptophan catabolism are used as precursors for the synthesis of other biomoleculesCatabolic pathways for phenylalanine and tyrosinePhenylketonuria (PKU)(Dr. Asborn Folling identif

53、ied the disease and Dr. George Jervis identified the enzyme): The inheritable metabolic defect was discovered in humans; It can also be caused by a defect in the enzyme that catalyzes the regeneration of tetrahydrobiopterin.phenylketonuria苯丙酮尿癥苯丙酮尿癥黑尿酸黑尿酸Alternative pathways for catabolism of phenyl

54、alanine in phenylketonuria 苯丙酮尿癥苯丙酮尿癥 Excrete in urine (“phenylketonuria”)(odor diagnosis)(odor diagnosis)苯乙酸苯乙酸苯基乳酸苯基乳酸 Role of tetrahydrobiopterin in the phenylalanine hydroxylase reactioncofactor Phenylalanine hydroxylase: also called phenylalanine-4-monooxygenase, belong to mixed- function oxida

55、se Catalyze simultaneous hydroxylation of a substrate by an oxygen atom of O2 and reduction of the other oxygen atom to H2O. Requires the cofactor tetrahydrobiopterin, which carries electrons from NADH to O2 and becomes oxidized to dihyrobiopterin in the process. It can be reduced by dihydrobiopteri

56、n reductase in a reaction that requires NADH. In humans, phenylalanine and tyrosine are normally converted to acetoacetyl-CoA and fumarate.Alkaptonuria (1897): - less serious than PKU - large amount of homogentisate are excreted and its oxidation turns the urine black. - Dr. Archibald Garrod made co

57、nnection between human disease and an enzyme. 黑尿酸癥黑尿酸癥酪氨酸血癥酪氨酸血癥5 AAs (five-carbon skeletons) are converted to -ketoglutarate4AAs are converted to succinyl-CoA(serine dehydratase)Methylmalonic Acidemia (MMA) Serine and threonine dehydratases are functionally and structurally related pyridoxal-phosph

58、ate dependent enzymes. L-serine dehydratase (EC:4.3.1.17) and D-serine dehydratase (EC:4.3.1.18) catalyse the dehydratation of L-serine (respectively D-serine) into ammonia and pyruvate. Threonine dehydratase (EC:4.3.1.19) (TDH) catalyses the dehydratation of threonine into -ketoglutarate and ammoni

59、a. 胱硫醚胱硫醚 甲基丙二酸血癥甲基丙二酸血癥白化病白化病黑尿酸癥黑尿酸癥苯丙酮尿癥苯丙酮尿癥甲基丙二酸血癥甲基丙二酸血癥槭糖尿癥槭糖尿癥高胱氨酸尿癥高胱氨酸尿癥Did a mother poison her son with antifreezer (ethylene glycol) ? The Patti Stallings caseAntifreezerMethylmalonic acidemia (MMA) A recessive genetic disorder (隱性遺傳病) of amino acid metabolism; MMA affects about 1 in 48,

60、000 newborns and presents symptoms almost identical with those caused by ethylene glycol poisoning. Methylmalonic acid, a breakdown product of the branched -chain amino acids isoleucine and valine, which accumulates in MMA patients .Dr. William S. Sly and Dr. James Shoemarker solved the murder myste

61、ry乙二醇乙二醇IS: 2,5-hexanedioneAnother confirmation experiment TheyTheyLeu, Ile, and Val are transaminated and decarboxylated by two common enzymes: branched-chain aminotransferase and branched-chain -keto acid dehydrogenase complex (being similar to pyruvate and -ketoglutarate dehydrogenase complexes)B

62、ranched-chain amino acids are not degraded in the liverMaple syrup urine disease: all three -keto acids and amino acids are accumulated (the urine of the patients smell like maple syrup)OxidativeOxidativeDecarboxylationDecarboxylation(E(E1 1, E, E2 2, and E, and E3 3) )Asparagine and Aspartate are c

63、onverted to oxaloacetateSummary Proteins are degraded to amino acids The turnover of cellular proteins is a regulated process requiring complex enzyme systems. The amino groups and carbon skeletons of amino acids take separate but interconnected pathways. Liver is the major site of amino acid degrad

64、ation in vertebrates. PLP facilitates the transamination and other transformations of amino acids. Glutamate collects and delivers free ammonia to the liver. Gln and Glu releases NH4+ in liver mitochondria. NH4+ in hepatocytes is converted into urea through the urea cycle in most terrestrial vertebr

65、ates for excretion. The conversion of ammonia to urea takes five (six) enzymatic steps. The rate of urea synthesis is controlled at two levels. The carbon skeletons of the amino acids are first converted into seven major metabolic intermediates. Some amino acids are converted to intermediates of cit

66、ric acid cycle by simple removal of the amino groups. Acetyl-CoA is formed from the degradation of many amino acids. O2 is used to break the aromatic rings of Pro, Phe and Tyr, as well as to oxidize Cys. Leu, Ile, and Val are degraded via reactions similar to fatty acid oxidation. A few genetic diseases are related to defects of Phe catabolism enzymes.