生物化學(xué)：chapter 22 biosynthesis of amino acids and nucleotides

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1、Chapter 22Chapter 22 Biosynthesis of amino acids Biosynthesis of amino acids and nucleotidesand nucleotides1. Sources of nitrogen, carbon skeletons and sulfur.2. The de novo and salvage pathways.3. Ways to balance the synthesis of each building blocks.For Dec. 25 and 27, 2012 (by Professor Zengyi Ch

2、ang)Key issues:Biosynthesis of Amino acids and nucleotides are closely related Nitrogen arises from common biological sources (N2 fixation). The two sets of pathways are extensively intertwined (with shared intermediates). Much common chemistry are found in both pathways: transfer of nitrogen (often

3、 from Gln) or one-carbon units (carried on tetrahydrofolate).The nitrogen in amino acids, purines, pyrimidines, and other biomolecules ultimately comes from atmospheric nitrogen, N2 (named as “azote,” meaning “without life”, by Lavoisier) N2 NH3 The amount of N2 newly fixed per year:By diazotrophic

4、(nitrogen-fixing) microorganisms, 60% (1011 kg); By Lightning and ultraviolet radiation, 15%; By industrial processes, 25%Unveiling the puzzle of nitrogen fixationJean L. Boussingault(1802-1887)Some plants raise thenitrogen content of the soil (1838) Hermann Hellriegel (1831-1895) and Martinus Beije

5、rinckand Martinus BeijerinckLeguminous plants fixnitrogen gas by itssymbiosis with Rhizobiumin the root nodules (1880s)James Carnahan and leonard Mortenson: cell-free extracts active in nitrogen fixation were obtained and nitrogenase isolated (1960s)Robert H. BurrisRobert H. Burris(1914-2010)(1914-2

6、010)15N labeled N2 enters NH3 and then Glu and Asp (1940s)Artturi Virtanen(1895-1973)Nobel Prize 1945N2 fixation is thermodynamically favorable, but kinetically extremely slowHas a bond energy of 930 kJ/mol(while that for a C-O is 350 kJ/mol)The chemistry of nitrogen fixation reaction Industrial (ir

7、on catalyst, 500C, 300 atm): Actual biological N2 fixation in diazotrophs:Required reaction:ATP hydrolysis is essential to reduce the heights of activation barriers along the reaction pathway, thus making it kinetically feasible. One of the most remarkable (expensive) reactions in biology!Nitrogen f

8、ixation is catalyzed Nitrogen fixation is catalyzed by the nitrogenase complex, by the nitrogenase complex, present only in certain present only in certain bacteria (diazotrophs like bacteria (diazotrophs like cyanobacteria and rhizobia) cyanobacteria and rhizobia) and energetically costly.and energ

9、etically costly. CyanobacteriaRhizobiaRhizobiaThe The nitrogenase nitrogenase complexcomplexNitrogenase (MoFe Protein)Nitrogenase (MoFe Protein)Nitrogenase reductaseNitrogenase reductase(Fe Protein)(Fe Protein)Electron Electron Donors:Donors:ferredoxin orferredoxin orflavodoxinflavodoxinATP binding

10、and ATP binding and hydrolysis hydrolysis is thought to both is thought to both drive the reduction drive the reduction of the P-cluster & of the P-cluster & to trigger a to trigger a conformationalconformationalchange in the change in the reductase reductase that causes it to that causes it to diss

11、ociate dissociate transiently transiently from the from the nitrogenase,nitrogenase,assuring assuring unidirectional unidirectional electron flow.electron flow.P clusterP clusterMoFe cofactorMoFe cofactor4Fe-4S cluster4Fe-4S clusterNN2 2NN2 2Ferredoxin was found to be a required for N2 fixation (196

12、2)MORTENSON, L. E. (1964). Ferredoxin requirement for N2-fixation by extracts of clostridium pasteurianum. Biochim. Biophys. Acta 81:473-477. Brown colored, most reducing agent Absolutely (together with ATP) required for N2-fixation by ex-tracts of C. pasteurianum (freed from ferredoxin by DEAE-cell

13、ulose treatment) when pyruvate serving as the electron donor.Also function in photosynthesis!The nitrogenase complex is The nitrogenase complex is extremely labile to Oextremely labile to O2 2 and various and various protective mechanisms have evolved: protective mechanisms have evolved: living anae

14、robically, forming thick living anaerobically, forming thick walls, uncoupling ewalls, uncoupling e- - transport from transport from ATP synthesis (entering OATP synthesis (entering O2 2 is used is used immediately) or being protected by immediately) or being protected by OO2 2-binding proteins (e.g

15、., -binding proteins (e.g., leghemoglobin).leghemoglobin). heterocystheterocystCyanobacteriaRhizobiaRhizobiaOO2 2 is believed to is believed todamage the damage the Fe-S centersFe-S centersAmmonia can be incorporated into biomolecules or converted to nitrite and nitrate, and back to N2 by certain ba

16、cteria(revealed in the 1980s)Substitute O2 asterminal electronacceptorReduced nitrogen in the Reduced nitrogen in the form of NHform of NH4 4+ + is first is first assimilated into Glu and assimilated into Glu and GlnGln(NH4+ is toxic at high levels to human beings and other mammals) Gln synthetase:

17、Gln synthetase: incorporates NHincorporates NH4 4+ + into into L-Glu to form L-GlnL-Glu to form L-Gln, , The The KKMM for NH for NH4 4+ + is low but is low but consumes ATPconsumes ATP Glu synthase: Glu synthase: An iron-sulfide flavoproteinAn iron-sulfide flavoprotein, , catalyzes catalyzes the red

18、uctivethe reductive amination of -ketoglutarate with theamination of -ketoglutarate with the use of glutamine as the nitrogen donoruse of glutamine as the nitrogen donor The sum: The sum: net synthesis of net synthesis of GluGlu from from a a-ketoglutarate and NH-ketoglutarate and NH4 4+ +! !Occurs

19、only in bacteria and plants.Occurs only in bacteria and plants.Containing FAD, FMN and Iron-sulfur centers.Gln synthetaseGln synthetaseGlu synthaseGlu synthaseGln synthetase-Glu synthase pathway of ammonia assimilationa a-ketoglutarate+ GluThe The Glutamine synthetaseGlutamine synthetase is a primar

20、y regulatory is a primary regulatory point in nitrogen point in nitrogen metabolism: being metabolism: being regulated by at least eight regulated by at least eight allosteric effectors and allosteric effectors and reversible adenylylation in reversible adenylylation in prokaryotes.prokaryotes. The

21、glutamine synthesis is constantly tailored to cellular needs!The The E. coliE. coli glutamine glutamine synthetasesynthetase: :12 subunits 12 subunits (dodecamers)(dodecamers)Active sitesat interfacesMg2+ Mn2+MnT Two rings of hexamerswo rings of hexamersMgMg2+2+ Mn Mn2+2+A bifunnel active site conta

22、inig two bivalent metal ionsA bifunnel active site containig two bivalent metal ionsThe The glutamineglutaminesynthetasesynthetase is is cumulativelycumulativelyinhibited by at inhibited by at least 8 allostericleast 8 allostericeffectors, mostly effectors, mostly end productsend productsof glutamin

23、eof glutaminemetabolism.metabolism.Each of the 50 kDa subunit containsbinding sites for all the 8 allostericeffectors in additionto the active sites!A striking demonstration of cumulative feedback inhibition and of control by a cascade of reversible covalent modificationsA specific Tyr in A specific

24、 Tyr in bacterial glutamine synthetasebacterial glutamine synthetase isis reversibly adenylylated by the catalysis of reversibly adenylylated by the catalysis of adenylyltransferaseadenylyltransferase, which is in turn is controlled by , which is in turn is controlled by reversible uridylylation.rev

25、ersible uridylylation.The animal The animal enzymeenzyme seems to be regulated by seems to be regulated by changing its oligomeric status (octameric to changing its oligomeric status (octameric to tetrameric).tetrameric). The inactive formAdenylylation increases the sensitivityAdenylylation increase

26、s the sensitivityof each subunit to the 8 allosteric inhibitors.of each subunit to the 8 allosteric inhibitors. AMPAMPTyrTyr397397The amide amino group of Gln The amide amino group of Gln supplies nitrogen to many supplies nitrogen to many other “acceptor” compounds other “acceptor” compounds via th

27、e catalyzes of via the catalyzes of glutamineglutamine amidotransferasesamidotransferases: : ammonia ammonia is generated first and then is generated first and then added to the acceptorsadded to the acceptors A proposed generalA proposed generalaction mechanism for action mechanism for glutamine gl

28、utamine amidotransferases.amidotransferases.Two-domain enzymesTwo-domain enzymesHighly conservedVariesL-Amino acid L-Amino acid biosynthesis: biosynthesis: t the carbon he carbon skeletons are derived skeletons are derived mainly from intermediates mainly from intermediates of of glycolysisglycolysi

29、s, , citric acid citric acid cyclecycle, and , and pentose pentose phosphate pathwayphosphate pathway; ;nitrogen from nitrogen from GlnGln and and GluGlu. . The stereochemistry at the -carbon atom is established by a transamination reaction involving pyridoxal phosphate and a conserved family of tra

30、nsaminases.The biosyntheses of the 20 amino acids use intermediates of the glycolytic cycle, citric acid cycle and the pentose phosphate pathwaysSix groups can be categorized123456Essential (indispensable) amino acids (William Rose, 1950s) Can not be synthesized de novo thus must be supplied in the

31、diet (humans). Mnemonic: VITAL pHTML VITAL pHTML (as designed by Zhilei Zhao)(as designed by Zhilei Zhao) A deficiency of even one amino acid will lead to negative nitrogen balance (more protein being degraded than synthesized) Nutritional quality of proteins for humans: Mammals fish & poultry fruit

32、s & plants.(1887-1985)Removal of Thr, but not His caused pronouncednegative nitrogen balance.(His found to be essential later)Pathways for synthesizing the “essential” amino acids are usually complex, involving 5-16 steps.T Transaminasesransaminases (pyridoxal phosphate, (pyridoxal phosphate, PLPPLP

33、) ) is key for amino acid biosynthesis mino acid biosynthesis aminamino acido acida a-keto acid-keto acidaminamino acido acida a-keto acid-keto acidA ping-pong mechanismALT(SGPT)AST(SGOT)Level of ASTand ALT arecommonly measuredin blood testreflecting liver orheart damages Stereospecificity of the St

34、ereospecificity of the transaminases: transaminases: addition of a proton addition of a proton from the Lys residue to one side of the from the Lys residue to one side of the quinonoid intermediate determines the quinonoid intermediate determines the L configuration.L configuration. Structures of tr

35、ansaminases are highly conserved.Structures of transaminases are highly conserved.Alexander Braunstein(1902-1986) A Russian scientistDiscovery of the transamination reaction (1937)Esmond E. Snell1914-2003)Discovery of the role ofpyridoxal (vitamin B6)in transaminases (1945)Discovery dates of the vit

36、amins 1913 Vitamin A (Retinol: retinal) 1910 Vitamin B1 (Thiamine; Thiamine pyrophosphate) 1920 Vitamin C (Ascorbic acid) 1920 Vitamin D (Calciferol) 1920 Vitamin B2 (Riboflavin; FAD, FMN) 1922 (Vitamin E) (Tocopherol) 1926 Vitamin B12 (Cobalamins; cobalamins) 1929 Vitamin K1 (Phylloquinone) 1931 Vi

37、tamin B5 (Pantothenic acid; Coenzyme A ) 1931 Vitamin B7 (Biotin; biotin) 1934 Vitamin B6 (Pyridoxine; Pyridoxal phosphate) 1936 Vitamin B3 (Niacin; NAD, NADP) 1941 Vitamin B9 (Folic acid; tetrahydrofolate) 1.1. Asp Asp, , Asn, Met Asn, Met, , ThrThr, and , and Lys Lys (also(also Ile Ile) ) are deri

38、ved from are derived from OxaloacetateOxaloacetate 2 steps2 steps8 steps steps2 steps2 steps4 steps4 stepsCysCysOne fourth of the One fourth of the building block amino building block amino acidsacids of proteins of proteins are are made here!made here! AspartatepyruvatepyruvateTetrahydrofolate (H4

39、folate) is a highly versatile carrier of activated one-carbon unitsThe one-carbon group, at threeoxidation states, is carried at N-5 or N-10 nitrogen atom (denoted as N5 and N10) or to both. H4 folate510Required for the de novo synthesis of purines, dTMP, and certain amino acids.2. Ala, Ile, Val, Le

40、u (and partially Lys) are derived from pyruvate4 steps3 steps1 step4 stepsThe nitrogen groupsare all provided byGlu!DehydratasePLP and TPP are twocofactors used here;The last four stepsof Ile and Val biosynthesisare catalyzed by thesame four enzymes;These three aminoacids seem to serveonly as buildi

41、ng blocksof proteins;The concept of allosteric control was largely developed here.ThrAla3.3. Glu, Glu, ProPro and and ArgArg are derived from are derived from a a- -ketoglutarateketoglutarate8 steps8 steps4 steps4 stepsGlu,Glu,Carbamoyl phosphateCarbamoyl phosphate ( (also for pyrimidine biosynthesi

42、salso for pyrimidine biosynthesis) )AspAspIn mammals, Arg can be derived via the urea cycle, thusIn mammals, Arg can be derived via the urea cycle, thusnot needed in adults (but essential for infants)not needed in adults (but essential for infants)(the (the a a-amino group is protected-amino group i

43、s protectedby acetylation for Arg synthesis)by acetylation for Arg synthesis)GluGluAspAspGluGlu4.4. Ser Ser, , GlyGly and and CysCys are derived from are derived from 3-phosphoglycerate3-phosphoglycerate3 steps3 steps1 step1 step2 steps2 stepsH4 folate, PLPIn mammals (Met provides Sulfur)In mammals

44、(Met provides Sulfur)PLPIn bacteriaIn bacteriaand Plantsand PlantsPLPb b-sythase-sythaseg g-lyase-lyaseSulfur assimilation,comparable toglutamine synthesisin nitrogen assimilation!5.5. Trp Trp, , PhePhe, and , and TyrTyr are derived from are derived from erythrose 4-phosphateerythrose 4-phosphate an

45、d and phosphoenolpyruvate (PEP)phosphoenolpyruvate (PEP)6 steps6 steps5 steps5 steps2 steps2 steps2 steps2 steps+ PEP+ PEP+ Gln, Ser + Gln, Ser PRPP PRPP + Glu+ Glu+ Glu+ GluIsozymesIsozymes; ;Feedback inhibitionFeedback inhibitionof 1st committed of 1st committed step;step;OperonOperon; ;Branching

46、pointsBranching points. .PEPPEP Branching Branching pointpointSerSerPRPPPRPP Branching Branching pointpointPEPPEPChorismateChorismatemutasemutaseGlnGlnThe The a a and and b b subunits of subunits of tryptophan synthasetryptophan synthase have have different enzymaticdifferent enzymaticactivities, ac

47、tivities, substrate substrate channelingchanneling occurs in this occurs in this enzyme.enzyme.a aa ab bb ba a2 2b2b2PLPPLP6.6. His His is derived from is derived from ribose 5-Pribose 5-P and and ATPATP8 steps8 steps+ Gln+ Gln+ Glu+ GluPurine nucleotide Purine nucleotide biosynthesisbiosynthesisInh

48、erently linked to the pathways of nucleotide formation;The beginning ofRNA to Protein Worldswitch? PRPPPRPPATPATPAmino acids are mainly derived from intermediates of glycolysis, the citric acid cycle, and the pentosephosphate pathway.VITAL pHTMLVITAL pHTML (as invented by Zhilei Zhao)(as invented by

49、 Zhilei Zhao)NHNH3 3(Gln)GluGluGluGluGlnGlnNHNH3 3NHNH3 3GlnGlnS S2-2-CysCysSources Sources of N, S of N, S in the in the amino amino acidsacidsAspAspGlnGlnThe rate of these biosynthetic The rate of these biosynthetic pathways are often regulated via pathways are often regulated via allosteric feedb

50、ack inhibition (and allosteric feedback inhibition (and at the gene level).at the gene level).A sparing effect of Ile on A sparing effect of Ile on Thr observed for a Thr observed for a threonineless mutant of threonineless mutant of E. E. colicoli; ;Ile found to be 100 timesIle found to be 100 time

51、sInhibitory than Leu to Inhibitory than Leu to inhibit Thr deamination.inhibit Thr deamination.End productThe first catalytic enzyme The first catalytic enzyme of Ile synthesis from Thr.of Ile synthesis from Thr.The first case of allosteric feedback inhibition:The first case of allosteric feedback i

52、nhibition:Thr dehydrataseThr dehydratase inhibited by inhibited by IleIle Umbarger, H. E. (1956) “Evidence for a negative-feedback mechanism in the biosynthesis of isoleucine”, Science 123, 848.Three common patterns of feedback inhibition in branching biosynthetic pathwaysConcerted Feedback Inhibiti

53、onConcerted Feedback Inhibition: Both I and F required to inhibit a.Cooperative InhibitionCooperative Inhibition: Either I or F weakly inhibits a; Cumulative InhibitionCumulative Inhibition:I and F each inhibits a to a given degree. Sequential feedback inhibition. F and I inhibit enzymes d and g, C

54、inhibits enzyme a Enzyme multiplicityEnzyme multiplicitya sensitive to I a sensitive to F. 1. Enzyme 1. Enzyme MultiplicityMultiplicity2. Concerted inhibition 2. Concerted inhibition (both Lys and Thr required)(both Lys and Thr required)A: aspartokinase isozymes(not present in animals!)Branching bio

55、synthetic pathways often coordinated via Branching biosynthetic pathways often coordinated via multilayers of feedback inhibitions multilayers of feedback inhibitions B: Homoserine dehydrogenaseC: Thr dehydrataseMany amino acids act in regulating the genes of their biosynthesis: trp and his operons

56、extensively studied.Amino acids are precursors of hormones, coenzymes, nucleotides, alkaloids, cell wall polymers, porphyrins (hemes), antibiotics, pigments, and neurotransmitters.GlutathioneGlutathione Glutathione S-transferase,GST, often used as aprotein tag for affinitypurificationArg, Gly, Met,

57、Glu, Cys used here.Messenger 15N and 14Clabeling (1940s David Shemin ) NO synthase(five cofactors)Synthesized in liver and transported to muscle. AntioxidantMelatonin (a neurohormone)“腦白金腦白金”Tyr, Glu, His, Trp serveas precursors of neurotransmitters, hormones, vasodilators. NAD+NeurotransmitterVasod

58、ilator Neurotransmitters and hormones 5-hydroxyltryptamine(5-HT)Inhibitory neurotrasmitter5-methoxy N-acetyltryptamineNucleotide biosynthesisThese bases are not intermediates! An ample supply of An ample supply of nucleotides is essential for nucleotides is essential for many life processes.many lif

59、e processes. NTP are precursors of nucleic acid synthesis. Source of energy (ATP, GTP). Nucleotide derivatives participate in biosynthetic processes (e.g., UDP-glucose in the formation of glycogen). Second messengers (cAMP and cGMP). Donor of phosphoryl groups (ATP, by protein kinases). Moiety of co

60、factors, NAD/NADP, FAD/FMN, CoA (ATP). Etc.Nucleotides are synthesized via either the de novo or salvage pathways.The base is synthesized from simpler starting materials A base is reattached to a ribose (ribonucleotides) (ribonucleotides) Isotope tracer experiments (mainly Isotope tracer experiments

61、 (mainly using using 1313C-,C-,1414C-labeled precursorsC-labeled precursors) in ) in pigeons (and later its cell free liver pigeons (and later its cell free liver extract) revealed the extract) revealed the originsorigins of the of the atoms in the purine and pyrimidine atoms in the purine and pyrim

62、idine rings (John Buchanan and Robert rings (John Buchanan and Robert Greenberg, 1946-51).Greenberg, 1946-51).Approach: Fed radiolabeled precursors to pigeons, Approach: Fed radiolabeled precursors to pigeons, and chemically degraded the uric acid they and chemically degraded the uric acid they excr

63、eted.excreted.BuchananBuchanan(1917-2007)(1917-2007)GreenbergGreenberg(1918-2005)(1918-2005)The purine ring is assembled on ribose-Pfrom simpleprecursors.(H4 folate)(H4 folate)123456789The pyrimidine ring is assembled from simple precursors before attached to ribose-P (or Gln)(or Gln)De novoDe novo

64、purine nucleotide purine nucleotide synthesis: the base synthesis: the base assembles on the ribose-P; assembles on the ribose-P; IMPIMP is the first nucleotide is the first nucleotide synthesized.synthesized.CommittingstepNonsequential steps 1, 3, 5 arecatalyzed by one multifunctionalprotein in euk

65、aryotes! (Unstable)1st ring closureStepwise assembly of the purine Stepwise assembly of the purine ring occurs on ribose phosphatering occurs on ribose phosphateRibose 5-PRibose 5-Pa a(remnant of ATP releasedduring His biosynthesis)Steps 10 and 11 are catalyzed by one protein.2nd ring closureAsp don

66、ates an amino group in a similar fashionas it does in the urea cycle.Biotin not used here !Biotin not used here !Biosynthesis of Biosynthesis of AMP & GMP from IMP.AMP & GMP from IMP.GTP as an energy source!Balanced synthesisBalanced synthesisof AMP and GMP:of AMP and GMP:AMP formation AMP formation requires GTP & requires GTP & GMP formationGMP formationrequires ATP!requires ATP!The biosynthesis of AMP The biosynthesis of AMP and GMP is regulated and GMP is regulated mainly by sequential mainly