BIOCHEMISTRY BIOC 441
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Date Created: 09/09/15
BIOCHEMISTRY 441 WINTER 2008 View the The DNA Story movie at one of the times posted on the course website Automated Online Video Screencasting is used in this course Screencast recordings of the classes are located at httpWWWcssWashingtoneduc0urseBIOC441A To watch a Screencast rightclick the Screencast link Adobe Flash Player is required Visit the Adobe Flash Player Download Center if Flash is not installed on your computer httpWWWadobecomproducts ashplayer 5 end c N G H ii CH DNA is a polymer of 2 NC 2 deoxyribonucleotides HOCH2 ill l2 25 NK H C ykNCH GCTAp 39o P o CH2 2 deoxyribose sugars Phosphodiester linkages Directional chain 5 to 3 I 4 Bases purines adenine amp guanine pyrimidines cytosine amp thymine g o E CH H39r 3 C CH I 0 N O P O CHZ NH2 ii 3 N N C A He I CH 0 c N G c H in CH RNA Is a polymer of 2 N N ribonucleotides 5 end HO39CHz in 4 1 K 3 II C GCUAp I OH 04 N 39o P o CH2 0 ll 5 if 11 U 0H IC CH ribose sugars N 39OPO CH2 NH2 Phosphodiester linkages g o I C A Directional chain 5 to 3 J H I g CH 4 Bases iquot H N N purines adenine amp guanine pyrimidines cytosine amp uracil RNA is easily hydrolyzed under alkaline conditions 39OIIDOCHZ N 39O OCHz o N o 1 0 39OPOCHZ N OH 0 o 0 H20 I POH of o 390 ha OH OH lt o I N N o Po CH2 HOCH2 mixture of 2 and u U o o o 3 monophosphate derivatives RNA 0 OH 0 OH 0Po oo shortened g g RNA The reaction proceeds through a 2 3 cyclic monophosphate intermediate Enzymatic hydrolysis of RNA by RNase proceeds through a similar intermediate Because DNA lacks the 2 OH group it is stable under alkaline conditions Why does DNA contain T rather than U Cytosine deaminates nonenzymatically to form uracil If this happens in DNA it constitutes a mutation Cells have a proof reading system that recognizes the error and replaces the U by C M ii N CCH H20 HNCltIH 04 NylH 04 NCH I cytosine I uracil Deamination of cytosine is of less consequence in RNA which is not the permanent repository of genetic information The phosphate groups of DNA and RNA are negatively charged A phosphodiester group has a pKa of about 1 and so will always be ionized and negatively charged under physiological conditions pH 7 Nucleic acids require counterions such as Mg2 polyamines histones or other proteins to balance this charge 1 39o hD o CHZ N Mquot39 0 1 P o CH2 N Mquot39 g 3 oPo HCO HoCH2 0H HCOH 0 5 HCOH HO39CHz N 0 9390 H OH HCOH Bfuranose CHon ring form I 0 ribose in its aldehydeform 0 ha 0 CH2 0 C2 exo The ring can adopt various puckered conformations in which C2 and C3 are in either exo or endo positions relative to the base and C5 The sugars are always in the Bfuranose cyclic form 0P03239 0H The nucleotide base can rotate with respect to the sugar The bases can adopt either syn or anti conformations but anti conformations are preferred HOCH2 O 0H 0H synAdenosine OH OH synCytosine c Nc HC NCN H Ho H20 0H 0H antiAdenosine HOCH2 o OH OH antiCytosine NH2 N The pattern of Xray diffraction by DNA fibers reveals a helical structure with steps of 34 and 34 A This x ray diffraction by calf mus NA wa measured by Franklin 8 Gosling In 952 The X pattern is indicative of a helix with a pitch of 34 A bottom reveal internal steps of 34 A Hbonds between WatsonCrick base pairs stabilize a double helix 0H HC Nc c c c H N I 3 G and C form q c Hquotquot 4quot Hb nd 3 o s G N50 N C Jq Huuf w H o IC Nc c c cCll3 I 3 A forms 2 Hbonds Pc H N H With T or U A N CM T Basepairing explains Chargaff s rules for the base composition of DNA A T G C It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material JD Watson amp FHC Crick Nature 171 737 1953 The Bform DNA helix has a diameter of about 20 A Base pairs fiii the center of the heiix the phosphates O are on the outside A base pair is more exposed to the soivent on one side the major groove at the top in these views than the other the minor groove bottom Bform DNA consists of a righthanded double helix with antiparallel strands 34 A per basepair 7 Fr L g major groove f 39 39 34A 10 bp per turn minor groove These dimensions are for DNA fibers In solution there are 105 basepairs per turn The antiparallel orientation of the two strands was demonstrated by synthesizing DNA enzymatically from labeled nucleoside triphosphates Kornberg synthesized labeled doublestranded DNA enzymatically from oc 32Plabeled 5 nucleoside triphosphates and hydrolyzed the DNA with a DNase that released the 3 mononucleotides For example with labeled ATP and unlabeled TTP GTP and GTP the sequence shown below gives pppA pppl DNA polymerase ng pfp39lgp p pcfpgpcip 3 3 z u r n l v x n 395 pppG pppC 3 pCprGpAprTpGpGpCp 5 H20 GpApCpTpApApCpCpGp DNase CpTpGpApTpTpGpGpCp In this example 24 of the Tp and 15 of the Gp are labeled What would you expect if the two strands were parallel Answer 14 of the Tp and 25 of the Gp would be labeled Highresolution structures of small DNA molecules have been obtained by xray crystallography and NMR NMR structure of a duplex DNA dodecamer in the DNAbinding sue of an interferon promoter J R Hulh et al Nature Slnlct Biol 4165711997 2eze pdb The two strands of the double helix separate reversibly at high temperatures The temperature at which this denaturation or melting occurs depends on the pH and salt concentration and increases with the GC content of the DNA The curves drawn here are schematic If the temperature is lowered the strands recombine The rate of recombination is inversely proportional to the complexity of the DNA Denatured 100 80 60 4O 20 4O 50 60 70 G0 I 7O 804 Temperature C 110 Doublestranded and singlestranded DNA differ in their optical absorption at 260 nm x 15 dA dG dU FA 10 E dc u s E a 5 A a l w 220 240 260 280 300 220 240 260 280 300 Wavelength nm Wavelength nm The conjugated n electron systems of the purine amp pyrimidine bases absorb strongly in the UV That s why UV light is mutagenic and carcinogenic The absorbance of doublestranded DNA dsDNA at 260 nm is less than that of either singlestranded DNA ssDNA or the free bases This is called hypochromism Hypochromism results from dipoledipole interactions between neighboring bases The excited states of an interacting pair of molecules can be described as linear combinations of the excited states of the individual molecules In certain geometries some of the absorption strength in the nearUV moves to bands at higher energies empty 7 L orbitals 9 8 strong 7 light Q absorption band 5 CD 5 E filled orbitals individual interacting individual base bases base strong band near weaker band strong band near 260 nm near 260 nm 260 nm Summary of the main structural features of Bform DNA Righthanded helix Two antiparallel strands held together by WatsonCrick hydrogen bonds Pitch repeat length 34 A 34 nm 36 rotation between residues Helix diameter of 20 A 20 nm Wide major groove narrow minor groove Chargaff s Rules A T G C Charged phosphates Bases in anti configuration The strands separate at high temperatures The solution structure is dynamic structures under DNA forms other 3dimensional some conditions Zform diamet DNA has a lefthanded helix with 12 bp perturn 18 A er and alternating syn and anti confer ations n probably occurs in We only in short stretches m T Schw 1841 11 quot Two views of the crystal strucrure of 539 TCGCGC in me DNAbinding domain of an RNAediting enzyme 63 anz elal Science 234 999 1qu pdb Hoogsteen pairs can stabilize triplex DNA structures 7 C H rotonated H o I I ll 39 b H H 2 NH 2 H HC Nc go H G C H H Nc c Hm 0 CH3 q c H lt 33 H Q C gtlH N 3 H N G C A T A protonated cytidine can form two H Thymidine can form two Hbonds bonds to the guanosine of a GC pair to the adenosine of an AT pair Tautomeric forms of G amp T can cause mutations due to mispairing during DNA replication K tsK I CH I ll CH 2N39CsNCxN 2N39CsNCxN The ring NH atoms of G and T have Guanine Guanine oKa values of keto form enol form about 9 At physiological pH about 99 of the 6 base is in the keto L form and 1 in the C CHa C CI39l3 enol form 393 EH 393 EH 0 N o N Thymine Thymine keto form enol form Palindromic sequences inverted repeats in DNA or RNA can form hairpin or cruciform structures 4i lava 3 7 7 mail if I is A Q E ETD 39i L 5 IIII3 3IIII 5 cruciform hairpin 5 3 IIII IIII Mirror repeats cannot form these structures I A palindrome reads the same in either direction Radar Madam I m Adam Cells contain a variety of types of RNA with different functions Principal kinds of RNA in E coli Mk l 133 5 33 Ei7iitjll liilleZX mRNA 6 25 25000 1000000 75 3000 2 tRNA 4 23000 30000 73 94 16 rRNA 5 35000 120 16 550000 1 542 82 23 1100000 2904 Eucaryotic cells contain an additional type small nuclear RNA snRNA Most RNA molecules consist of a single strand that folds back on itself to form doublehelical regions hair in S39ngle internal F2 strands loop V A A 0 GO 0 P O 7 T In RNA G pairs with C The loops and and A pairs with U hairpins have few or no basepairs 111e 3dimensional structures of even relatively small RNA molecules can be very complex tube licorice representation representation S1ructure of a hammerhead rihozyme Ribozymes are RNA mulecules that catalyze events in RNA chains A amp B A 1mmepdb Some singlenucleotide polymorphisms in a gene probably change the secondary structure Ihe LPS and the LPS LPS quotl Ap s mas AGA200N5MII AGJBQXC HW GLENamid DNA base sequences can be determined by using DNA polymerase and dideoxynucleotides Synthesize DNA enzymatically in the presence of a small amount of a dideoxynucleotide eg ddTTP and larger amounts of ordinary ATP GTP CTP and TTP using a primer and one strand of the DNA to be sequenced as a template DNA polymerase synthesizes the new strand in the 5 a 3 direction The dideoxynucleotide interrupts DNA synthesis wherever it is incorporated This happens at random points where the corresponding base occurs in the sequence In this illustration each DNA fragment ends in ddT which is labeled with a red dye o 039 039 O O 5 primer 3 synthesiS gt ddTTP GCCGCATGTCGTAACTW ICGGCGTACAGCATTGAA 3 template 5 continued DNA sequencing can be automated Repeat the procedure with ddC ddA and ddG using a different fluorescent dye to tag the dideoxynucleotide in each case Then mix all the DNA fragments and separate them according to size by electrophoresis Separating the DNA fragments gives a ladder of bands each ending in a particular dideoxynucleotide at its 3 end and tagged with the corresponding dye This procedure has been automated in machines that can sequence more than 3x106 bases in a month GCCGCATGTCGTAACTT 5 primer 3 synthesis gt GCCGCATGTCGTAACTT EGGCGTACAGCATTGAM template 5 02 amino acids amp Nitrogen cycles betweenoxidized other organic amp reduced forms in the biosphere compounds synthesis degradation reduction microorganisms animals amp plants amp some plants amp animals microorganisms anaerobic bacteria denitrification 39I39tr igequot fixation nitrate i q gt gt ammonium 39 anaeroblc Rhizobium amp baCteria some other bacteria n ca on n ca on Nitrobacter amp Nitrosomonas amp other soil bacteria nitrite other soil bacteria more oxidized more reduced In the industrial Haber process N2 is reduced to NH3 by H2 at high temperature and pressure with an iron oxide catalyst 2 NH3 The reaction is exothermic by 924 kJmol at standard temperature amp pressure but has a very high activation energy The roots of leguminous plants have nodules that contain N2fixing bacteria Bacteroids rodlike bacteria containing nitrogenase live inside the nodule cells nodule cell nucleus bacteroids Nitrogenase is very sensitive to 02 It is protected in the nodules by leghemoglobin a heme protein with a strong affinity for O2 Leghemoglobin is produced by the plant but carries 02 for reduction by the bacterial respiratory chain keeping the 02 concentration low l l This electron micrograph is colorized artificially 2 um Nitrogenase from Azobacter vinelandii has ironsu u and ironmolybdenum centers Azolobacler are MoFeS cluster Mo7Fe95 8Fe7S cluster Fe protein 39 4Fe4S cluster Mg ADP 2 Two ATPbinding sites structurally ins 1n2c pdb homologous to Gprole Cys residue of the protein The FeMo cofactor contains homocitrate The mechanism ofthe N2 lixation reaction is not known but intermediates in which partially reduced derivatives of Fe N2 replace one ofthe O atoms S bound to the Mo have been proposed Mo 0 C Homocitrate Cihydrony carboxyadipic acid lwon t expect co to remember this structure Nitroenase uses 8 4 c ASH 4 302 electrons and 16 ATP 4 pyruvate 4 acetyICOA to reduce N2 2 H to 2 NH4 H2 8 ferredoxin reduced The Fe protein transfers one electron at a time to the FeMo protein 8 Fe protein reduced 8 FeMo protein reduced The ATP stoichiometry is uncertain Only8 2 NH4 N2 ATP are needed under some conditions H2 2 H The first step in catabolism of most amino acids is transamination 02 H3NCH co amino acid II II ocketo gt lt acid 00239 3930239 420 H3Nl3H 9H2 9H2 9H2 9H2 002 002 glutamate aketoglutarate The main function of transamination is to funnel amino groups into a small number of amino acids particularly Glu amp Asp Some amino transferases transaminases are specific for ocketoglutarate and Glu others use oxaloacetate and Asp Transaminases use pyridoxal phosphate as a prosthetic group CH20 H20 H L Ho CH3 H20 pyridoxa phosphate Pyridoxal phosphate forms a Schiffbase aldimine bond to a lysine residue of the enzyme This reaction is readily reversible CH20 CH20H HIllCH NH HOCH2 NH Cl 39 3 2 Ho CH3 Ho CH3 pyridoxamihe phosphate pyridoxine hydrochloride vitamin Be Pyridoxal phosphate transfers the amino group by shuttling between aldehyde and amine forms H amino 39OZC I3 R1 39OZC C R1 aketo acid 1 IH3 acid 1 Ho CH20 HO CH2 pyridoxal I pyridoxamine phosphate CH3 N CH3 N phosphate on enzyme H H on enzyme H gt lt I acid 2 NH 393 acid 2 Both steps occur with the coenzyme bound noncovalently to the enzyme This is a classic pingpong enzyme mechanism The positive charge of the pyridoxine ring facilitates Film interconversions of Schiffbase intermediates acl H quot I E 2 H H OZCCR H20 m S h I H 39o Catc R 39ff NH2 02C I R 2 I base NH N CHo H CHOH Ho CH20 Ho CH20 Ho I pyridoxal CH3 phosphate H3 CH3 otketo 39ozcc R acid H H20 Ozc39 39R i CHzgin Ll CH Ho CH20 Ho CH20 CH3 N pyridoxamine CH N H phosphate H base 3 H The active site has additional residues that could facilitate proton binding and release Echiff base formed from P39LP amp 2me thyIAsp aspartate aminotransferase 1ajspdb Related enzymes use pyridoxal phosphate to catalyze amino acid racemizations and decarboxylations o 391 9 I I 39I39 39ogcrc R 322 ozc c R H20 co2 amino NH2 lt acid H c R II CHO 39N CH HO CH20 HO H 39 CH3 m H c R N CH3 N ll H CH amlne H HO CH20 39 H20 1 H 393 R H NH2 CH3 m Schiff Amino acid decarboxylases generate amines that serve as neurotransmitters CO 39 C COZ39 I 2 3902 I H3N39C39H H3NCH H3Nl3H EH I CH2 2 5hydroxyTrp H2 Glu l dihydroxyPhe EH2 DOPA Ho NH 00239 OH OH CO v1 C02 J C02 V4 2 NH3 NH3 NH3 I I I CH2 CH2 CH2 I l I I3H2 CH2 CH2 EH2 HO 00239 0H NH yaminobutyrate OH I GABA dopamine serotonin Also Histidine gt histamine 002 IIIIIIIII v ingested cellular protein The amino roups of lutamic acid and lutamine can be released as ammonia in liver mitochondria prevent it Terrestrial organisms must protein 5 igoz39 transaminases i702 H NCH co aketo amIno 3 I I 39d adds R R acI s i 239 2 Io H3NCI3H CH2 PH 0Lketo l 2 quot Glu glutarate H2 glutamate IH2 CO 39 CCgt2 dehydrogenase 302 2 H3NltI3H NADH or 9H2 NADPH H NAD 3r H20 I3H2 NADP CONHZ But ammonia is toxic particularly to neural tissue W firstng other tissue from accumulating Ammonia is incorporated into many biological molecules through glutamine and glutamate CO 39 CO 39 CO 39 I 2 m 2 W4ATP ADPPi I 2 I3 H3NI3H H3NI3H CH2 CH2 7 I3H2 I I CH2 ICHZ H20 H2 Gln NADH NAD or C02 or NADP 02 G39quot o H2 NADPH ocketo k 3 J I t t 1 g u ara e F 239 F 239 wCH 0 ocketo Glutamate dehydrogenase 1 EH CH2 QIUtarate and glutamine synthetase 2 I 2 I are found in all organisms CH2 CH2 Reaction 3 occurs in plants amp em I bacteria but not animals C02quot C02 Glutamine serves as a donor of amine groups for synthesis of many other molecules carbamoylphosphate glucosamine6P gt glycine histidine h In most terrestrial animals Gln also tryptop a carries ammonia in the blood to the AMP liver amp kidneys for excretion as urea CTP Glutamine synthetase catalyzes formation of glutamine from glutamate and NH4 glutamine synthetase CO 30 I 2 0 H3NCH H3N39C39H H3NCH ATP ADP Cquot392 NH4 Pi I EH2 I CH2 CH l3H2 LA I 2 J EH2 COl co o4 CNH Glu 2 0 2 Gln The reaction proceeds through an enzyme bound yglutamylphosphate intermediate NH4 H20 glutaminase In terrestrial animals Gln carries ammonia in the blood to the liver amp kidneys where it is hydrolyzed for excretion as urea Glutamine synthetase is inhibited allosterically by many of the endproducts carbamoyl IIIIIIIIIIIIIII n Glu glutamine Gln phosphate E CO2 synthetase gigcosamine ATP ADP H3NiH NH4 Pi alanine ltH2 U f 39 4 A A A 4 9 I C02 5 5 5 quotNH histidine Theinhibitory tryptophan effectofallthe productsacting ch AMP togetheris E E E E i 5 greaterthanthe 5 E i sumoftheir f 1 i 39 individual effects 5 E coli glutamine synthetase also is controlled by covalent modification uuuuuuu Gln ATP PP glutamine synthetase inactive OH OAMP deadenylylation V G ADP P aketo d I o a en l39OU 39s glutarate 39 y g p 39 0 ll 0 I39D OCH2SZAdenine 0 o The regulation by Gin and ocketoglutarate involves similar covalent modifications uridylylation of the enzymes that add or remove the adenyl group to glutamine synthetase Bacterial glutamine synthetase has 12 identical subunits SET 31 fquot39 V t Views of the Salmonella typhimurium enz me parallel and perpendicular to the 6fold symmetry axis 2glspdb Alanine carries amino groups from muscle to the liver for excretion 273 o l HzNCNH2 1 glucose glucose urea amino acids l NH Glu 302 302 CO 3 0 I Glu CH pyruvate CH3 3 pyruvate 02 aketo39 H N39C39H H NCH glutarate 3 Ala 3 0c keto K CH3 Ala 3H3 glutarate Synthesizing carbohydrates from CO2 and water presents a formidable thermodynamic problem 6 CO2 6 H20 gt CEHuOIi 6 02 AG 3 679 kcalllnol 2480 kJmol K 11139 6 Photosynthetic organisms use the energy of light to driv carbohydrate synthesis against this enormous gradient The energy of red light 700 nm is E MIN 41 kcalleinsteinquot 172 kJIeinstein s co2 6 H10 c nuoa s 02 46quot 4290 kcallmol 6398 kJImol K 10quot2 i 39 39 39 39 fphotons N M n Planck s constant ammo34 us v frequency squot When light raises a molecule to an excited electronic state the molecule becomes a stronger reductant f LUMO g LUMO E m HOMO a is u E Home LIGHT gt E electrons A B A B The photochemically reactive pigments are chlorins or bacteriochlorins which are structurally related to hemes Hemes Chlorophylls Bacteriochlorophylls symmetrical asymmetrical more asymmetrical 1 7 systems 7 systems ys ms absorb blue absorb blue absorb blue orange light a red light amp nearIR light The photochemical reactions of photosynthesis take place in integral membrane protein thylakoid outer membrane lumen inner membrane stroma Most of the pigments in photosynthetic cells do not participate in the electrontransfer reactions of photosynthesis Instea they serve as an antenna that increases the absorption of light At high light intensity the 39 re R Emerson A W Arnold measured the amount of Oz maxmum 0 Based per ash formed when they excited algae 1 with short ashes of light was aha 1 0 per 2400 c 539 00004 021cm 00002 At law light intensity 1 02 is formed for Veach a photons 0 absorbed yellow dashed line 0 0004 was Light absorbed photonsCm The antenna system of purple photosynthetic bacteria has circular pigmentprotein complexes The LH1 complex with 31 transmemhrane xihelical peptides yellow and 30 BChls K colors surround 9 reaction b own where the ions occur een electrontransfer real A w Roszak el al Science 302 1965 2003 1pyhpdb W en the antenna is excited with light excitations are transferred to the reaction center within Smalle LHZ antenna omplexe transfer energy rapidly to LH1 view normal to antenna BChIs are the membrane green and blue in this gure 1 p5 103912 s The LHCll lightharvesting complex ll antenna protein of plants is h l h b I a trimer with 14 chlorophylls and 3 carotenoids per protein subunit T E e ectmquot Gamers mt 9 ad reamquot center are arranged around an axis of approximate rotational symmetry r 5 e 5cm dimer Two of the our BChls 1 a form a dimer Pam 17 Bohl 39K e ectron donor BChI bacteriochlorophyll Axis approximate BFh bacterlopheophytln rotational symmetry BChl with 2 H ln place oiling Z Liu etal Nature 425 257 2004 1rwlpdb Q ubiqulnone The sequence and kinetics of the initial electrontransfer reactions can be studied by exciting RCs with short pulses of light The reaction center of purple photosynthetic bacteria has 3 to 4 subunits depending on the species The two central subunits are integral membrane 39ns 39th protel WI homologous structures baa train or short pulses splitter A small number of hacteriochlorophylls and other electron carriers are bound to the proteins vary the path 4amp7 length for the probe pulses 1 and 12 different detect di eren electron carriers Ahsnrhance change measure the intensity view parallel to of the transmitted 2 mm H Delay between excitation and probe pulses over many pulses When the complex is excited with light an electron move from the BChI dimer 1 3 F870 to a BFh and then to a quinone BChl dimer f v UV 3 ps The photosynthetic electrontransfer system i P870gt Bchl BPh Light n purple bacteria is cyclic DA 1 Reduced uhiquinone Qstl a 1 dissociates from e RC and 39 diffuses to the cytochrome cc cytochrome be complex where it is reoxidized cytochrome c P870 2 Reduced cytochrome c diffuses back to the RC to complete the cycle Electron flow pumps protons across the membrane Plants have two photosystems P700 that work in series to move electrons from water to NADP lt1 PGBOK FeS centers Phe fe rredoxin Light NADPH Liam PEA PQE NADP Cyt b f complex Photosystem I 02 plastocyanin quot1V P700 Mn center Photosystem ll PQA P03 piastoquinone m P680 piastocyanin a Cu protein terreooxin an FeS protein The reaction center of Photosystem II has 20 subunits it binds 32 molecules of Chla green Most of these are part of the antenna KN Felreira et al Science 3031331 2004 155Lpdb B LDII el al Nature 435 104012005 Zaxlpdb The core of the Photosystem ll reaction center The Fhotosystem I reaction center has 12 subunits Is very simllar to that of purple bacteria It binds abnll 1 molecules otChl Those shown in yalluw are part oiths antenna lsslpclb 2axlpdb The arrangement of the Chi electron carriers blue agaln is very simllar to that in bacterial s ammo vlaw normal to embrana Electron carriers in the Photosystem ll reaction center Two of the FeS centers in Photosystem I are in subunit C on the stromal side of the membrane E333 E354 CPA H332 H331 lk l 7139 PsaC I 3 r r PsaE W 39 Cterm 0341 The cluster of4 Mn atoms and a betwee Pushkar at all Proc Natl Acad Sci USA 705 1879 2008 n the Mn I ustera on site and is unique viaw parallel to the membrane surface Plants have two photosystems P700 that work in series to mov electrons from water to NADP h39 3 1 PGEO r FeS centers Phe Ferredoxin nght P P NADP NADPH Lighl 0 Q Cyt b f complex Photosystem I plastocyanin quot2V P700 Mn center Photosystem ll Pom P03 plastoquinone Q phyloquinone P680 lasla anin aCu protein rerredoxin a soluble FeS protein Experimemal 1 The efficiency of 01 evolution drops at evidenca for w above 680 nm but can be me Z scheme increased by green background light a lt 3 o 1 2 In WIth background Ilght 02 released Man absorbed wlthout background ght 630 700 720 Wavelength nm N The green background light has this quotW50quot 339va 3quot effect even if it is turned off 39ust Am SCquot USA 3 m 1957 before the red light is turned on Zscheme can explain these Pm r th The results if only one o o photosystems absorbs far red light PSBO Photosystsm l Photosystem ll H10 P700 Measurements of partial reactions eg P700 photooxidation showed that both photosystems absorb below 680 nm but P53 only PS I absorbs at longer wavelengths 2 Green light can either WOT oxidize or reduce components between PS ll amp PS I but far red P680 light only oxidizes them h PSI NADP quot28 PS ll P700 L Duysens amp 1 Amesz Blocmm P680 Blophys Am 64 243 1962 cyt f r oxidation time gt 3 Farred background light decreases fluorescence from PS ll PTDO NADP PS II antenna 11 I lquot l FS ll complexes are located mainly in stacked regions of the thylakoid membrane PS I complexes are mainly in unstacked lamellae ATP synthase stacked membranes granal membranes unstacked membranes stromal amellae LHClI The LHCll antenna complex can move between these two regions Phosphorylation causes the LHCII antenna complex to move to unstacked regions 8 associate with PS l Thls increases the fractlon the absorbed light energy u t f of 39 39 M thatgoes toPSI all pm Ir J gnaw will 39 3 OH 1 1 I 7 l lill llllfclll lw 5 llllllllallglllll t l l l l V 1 ll l w The kinase that phosphorylates P700 LHCll is activated when the ratio PQHZMPQ i hi h re ecting overexcitation of photosystem II P680 Light 0H2 NADP p A 939 Photosystem l H20 P700 Photosystem II LHC u then moves to PSI improving the balance of P630 excitation of the two systems 02 evolution requires accumulation of four oxidizing equivalents ZHZO gt oz4ev4H 02 evolved per flash r 1 16 20 24 flash number Each step removes one electron from the Mn04ca complex A plausible mechanism e of O2 evolu in a e i Ca XL H 5 2 Mn HI 0 He e 2 EM Sprnviero et a1 JACS 130 3428 2008 54 S3 The chloroplast cytochrome b5 complex is homologous to the bc complexes of mitochondria and photosynthetic bacteria cytochrome b6 wlth two hemes red Ch green amp carotanoio orange found only in baf thylakold membrane quinone plastoquinone in bar ubiquinone in mitochondria cytochrome bslis a homodimar with multiple subunits H Zhang et al Proc Natl Acad Sci 100 5160 2003 1vl5pdb Oxidation oi PQH2 by the cytochrome b5 complex pumps protons across the thylakoid membrane plastocyanin thylakoid lumen thylakold membrane stromal space Proton pumping generates an electrochemical potentlal gradient that is used to make ATP The 0 cyclequot enables the cytbsf complex to pump 4 protons for each QH2 that is oxidized plastocyanin thylakoid lumen thylakoid membrane stromal space The mitochondrial E bacterial cytbc complexes also carry out a 0 cycle Proton and electron circuits in chloroplasts The electrontransfer reactions driven by light acidify the thylkoid lumen relative to the stroma MWWWWWWWW WWIA r 000000000000000000000 l N ADP NADPH r r r r r r r r r r r r r r r r r r r r r 1 h Ir I 4444949 11 44 54f li gli W 444949 f quot quot c 7 WI47179 4 r myiuyozyoy 4 44949 421 quot 4 4 W 40Q 3 r 44 e m 39 4 4 4 4 49 I 1444 1 I 499999919919999949 4 tr ma l 4 4 11 919999 4 9444444444 74quot 399 394 9 9 9 U s 44 4144 4444 in rw r Irr 4 4 4 4 44 4 4 4 4 29 lr A P4 4 4 4 4 4 4 44 9 9 4 4 o o oV p xix9 97111111494 Isaquottiles 9111411 The ATP synthases of chloroplasts and photosynthetic bacteria are very similar to those of mitochondria and nonphotosynthetic bacteria In ner membrane ATP synthase The orientation of the ATP synthase relative to the electrochemical potential gradient generated by electron transfer is the same in all cas s inner 7 lhylaknid membrane membrane intermembrane fhvlakoid pace 5 lumen Mitochondrion chloroplast Please v w the he DNA Story mo atone oflhe times pasted on the course website H l DNA is a polymer of z NCxu 2 deoxyribonucleotides c GCTAp c CH 239deoxyribose sugars I 0 N Phosphodiester linkages F o EHZ Directional chain 539 lo 339 if lclquotMCH A 4 Bases Hc NCN purines adenine 8 guanine pyrimidines cytosine 8 thymine 339 and DFOIZ39 c c quot G H n RNA IS a polymer of Z 5 ribonucleotides 5 end HUEH 4 C GCUAp o O CH an i i U 0quot 9c CH ribose sugars I 0 N oP o cH MHZ Phosphodlester linkages 0 N Directional chain 539 to 339 HE quot CH 4 Bases H NCN purines adenine amp guanine UEO CH1 o pyrimidines cytosine s uracil a 339 end DFO32 0quot RNA is easily hydrolyzed under alkaline conditions D 6 OPOCH N U39F OCHx 6 I II o I 39DFO CH 391 1 0 D o gt OH P Hzo I s our er a 3 r W 1 o t N 4 N 0 P 0 HocHl mixture of 239 and H u 0 Ho 0 o 3 monophosphate derivatives RNA 0 DH 0 OH Uo 0L0 shonened y g RNA 39 nemleuiate RNA U Because DNA lacks the 239OH group it is stable under alkaline conditions Why does DNA contain T rather than U Cytosine deaminates nonenzymatically to form uracil lfthis happens in DNA it constitutes a mutation Cells have a proof le 9 C N CH H20 Hit05in will lmLu I cytosine I uracil Deamination of cytosine is of less consequence in RNA which is not the permanent repository of genetic information The phosphate groups 5 are HocH N negatively charged 0 I M D ltla o c lo N o A phosphodiester group has a pKa of aboutl no so will always be ionized and negatively M Charged under physiological conditions pH 7 Nucleic acids require counterions ch 5 M 2 polyamines histones or other proteins to balance his charge Hc0 HoIznZ on o Hcon HOH YO H OH quotxiOH pruranase CHIDH rlngitorm rlhose in us aldehyde form The ring can adopt various puckered conformations 39 e in either exo or endo in which C239 and 0339 ar positions relative to the base a The nucleotide base can rotate With respect to the sugar The bases can adopt either syn or anti conformations ndCS on on synAdenosine on on syncytosine 5 The HOCHl N the 5 o sugars are always in furanose cyclic form 3 oPogz OH on on antivAdenosine OH on antivcytosine The pattern of Xray diffraction by DNA bers reveals a helical structure with steps of 34 and 34 A l N This xray diffraction by calf thymus DNA was measured by Franklin amp l Gosling in 1952 The X a bottom reveal internal steps of 34 A Hbonds between WatsonCrick base pairs stabilize a double helix 393 quot 39 quot uNHATGC immediately suggests a possible copying mechanism for the genetic materialquot JD Watson amp FHC Crick Nature 171 737 1353 The Bform DNA helix has a diameter of about 20 A 39 of the helix 1 out me A base pair is more exposed to the solvent on one side the major groovequot at the top in these views than the other the minor groove bottom Bform DNA consists of a righthanded double helix with antiparallel strands 34 A per basepair major groove 34 A 10 hp per turn minor groove These dimensions are for DNA bers in solution there are 105 basepairs per turn The antiparallel orientation of the two strands was demonstrated by synthes ng DNA enzymatically from labeled nucleoside triphosphates 32Plabeled 539 nucieuside lnphusphates and hydrolyzed the DNA with a DNase lhat released the 339 mnnonuclenlides For example with iabeled ATP and unlabeied 39I39I39P GTP and CTF the sequence Shown below gives pppA ppml DNA polymerase 539 PGPAPCPTPAPAPCPCPGP pppG mac 39 nCnTpGpAprTpGpGpCp WGptAptCptTpApApCpCpGp DNase tCpTpGpApTpTpGpGpCp In this example 24 of the Tp and 15 of the Gp are labeled What would you expect lithe t I 4 of the l Highresolution structures of small DNA molecules have been obtained by xray crystallography and NMR NMR structure of a duplex DNA dodeoamer in the DNAbinding site of an interferon promotor J R Huth el 3L Name Smurf Biol 4 657 1997 2ezepdb The structure le is available at wwwresborgpdbhome The two strands of the double helix separate reversibly at high temperatures The curves lf the temperature is lowered the strands recombine The rate of recombination is inversely proportional to the complexity of the DNA quotn Denatured 40 50 60 70 GO 30 90 100 Temperature DC Doublestranded and singlestranded DNA differ in their optical absorption at 260 nm 15 l l dA as nucleotides dU SSDNA quot 1n dc dsDNA E s 5 5 2 n m a 392 n 260 230 m 260 Wavelength tum m sun Wavelength nm The conjugated xelectron systems of the purine K pyrimidine bases absorb strongly in the UV That39s h V light is mutagenic and carcinogenic The absorbance of doublestranded DNA dsDNA at 260 nm is less than that of either singlestranded DNA ssDNA or the free bases This is called hypochromism Hypochromism results from dipoledipole interactions between neighboring bases combinations of some Ul 39ui eneigie empty r orbitals gt 9 strong a 5 quot9 Q absorption 2 band 3 e u E lled orbitals individual interacting individual base bases base strong band near weaker band strong band near 260 nm near 290 nm 260 nm Summary of the main structural features of Bform DNA Right handed helix Two antiparallel strands held together by WatsonCrick hydrogen bonds Pitoh repeat length 34 A 34 nm 3936 rotation between residues Helix diameter of 20 A 20 nm 39Vl de major groove narrow minor groove 39Chargaff39s Rules A T G 39Charged phosphates Bases in anti con guration The strands separate at high temperatures 39The solution structure is dynamic DNA forms other 3dimensional structures under some conditions 5 on 3 mations I probably occurs in vivo only in short stretches Two views of the crystal 1 structure of 539 139CGCGCG3392 39 the DNA inding domain of an RNAediting enzyme 19411999 1qupun y Hoogsteen pairsquot can stabilize triplex DNA structures Thymidine can form two Hbonds to the adenosine of an AT pair A protonated cytidine can form two H bonds to the guanosine ofa G C pair V T Schwartz et al Science 234 Tautomeric forms of G amp T can cause mutations e to mispairing during DNA replication H quot c H Nl 3 CH H N I u an I N N I Ncm The nng NH atoms of G and T have Guani uanine pKa values of enol form about 9 At ne keto form physiological pH b CH CH form and 1 in the c c enol form 4 uc NCH c CH Thymine Thymine keto form enol form Falindromic sequences inverted repeats in DNA or RNA can form hairpin or cruciform structures cruciform hairpin 539 Mirror repeats cannot form these structures Radar Madam l39m Adam 39A palindrome reads the same in either direction Cells contain a variety of types of RNA with different functions Principal kinds of RNA in E coli mRNA 6 v 25 25000 1000000 75 3000 2 RNA 4 23000 30000 73 94 16 RNA 5 35000 0 16 550000 1542 82 23 1100000 2904 Eucaryolic cells contain an additionai type sman nuclear RNA snRNA Most RNA molecules consist of a single strand that folds back on itself to form doublehelical regions internal strands hairpin P C w h C and A palrs WIth U or no basepairs The 3dimensional structures of even relatively small RNA molecules can be very complex licorice represenlation Structure of a hammerhead ribozyme chains A amp B immelpdh m m um um SinglequotUCIEOWB 122232 5 3 Nita polymorphisms m a gene can change i com r17lt17gt secondary structure of Naplalypn 5MP the mRNA LPS 5 c s a J m A r c M means m A c c y m u LPS and the LPS m E HPS A5 725 Maimoi A6 ae caim DNA base sequences can be determined by using DNA polymerase and dideoxynucleotides GTP CTP This 1quot 1quot 1 D P OP OP O H1 II o o o o 539 primer 339 synthesis ddTTp CATGTCGTAACTT CGGCGTACAGCATTGAA 339 lemplaie 539 cuminued DNA sequencing can be automated Repeat the procedure with ddC MA and ddG using a different fluorescent dye to each case Then mix all the mm GCCGCATGTCGTAACTT Jllllllllllllll electrophoresis Electrophoresis of the DNA fragments gives a ladder of bands each ending in a particular dideoxynuoleotide at its 39 nd and ta ed with the corresponding dye This procedure has been 3x105 bases in a month ll 539 primer 339 synthesis CATGTCGTAACTT CGGCGTACAGCATTGAA 39 template 539 Plants and bacteria synthesize aromatic amino phosphoenol pyruvate 02 902 I P 0 pi 0 c 1 I z cuou IIHO FHDH NAD 9Hon EHOH NADH 3quoton culo cuzo erythrosent phosphate 0H lto2 029 ed L phenylpyruvate Hoquot H prephenate acids from carbohydrates via shikimic acid shikimale HO co 7 quot20 H glyphosate m C cuNHcuo ADP EDI 20 I CH2 cozr p 12 t Hoquot H chorismale I don39t expect you in Phenylalanine and tyrosine break clown to fumarate and acetoacetate 02 NADH quot0 NAD Ph 9 quot Nquot T e CHZCHCOZ HO CHICHCoi y39 phenylalanme h dro lase y W D akelaglularate hamogentisil 0quot no 02 539quot acid 395 1 Q HzCOZ39 HOQCHzCCOZ phydmxyphenylpyruvate Ho dioxygenase p hydmxy39 phenylpyruvate 02 homogentisate H 12ltdioxygenase fumarylaceloacetale v 5 9 o G H 39OzCCCCCH2ECHzC01 oc maleylacelaacetate CHIIbCHZCoz39 o acetoaceiate I won39t expect you to knmw the A e coz CH35CH2coz names oflhe H iumarale occ c enzym other details of reactions 36 Genetic defects in phenylalanine and tyrosine catabolism can cause several disorders 02 NADH H20 NAD l phenylalzn ne l ymsine phenylalanine hydroxylase 7 quot95 amln 5 ranslerase Glu W 02 02 l 39 L i phydroxyphenylpyruvale dioxygenase homogentisale 12dinxygenase 9 l i Hz was met rsl 1 Z lumarylaceloacetase Alk an inherited disease ma 7 I Wai39quotked 539quot939e l lumarale l acetoacelate l W en ymeGarrod 1900 77 ap nu Individuals with phenylketonuria convert phenylalanine to products other than tyrosine 02 NADH H20 NAD v Phe NH NH Tyr CHzCHcoz 34 HOQ CHZCHCDZ39 pyruvale blocked in PKU Ala 0 Q cuzEcoz phenylpyruvate lflhe disorder is H20 NADH NAD 02 OH Q CHI60239 Q CHzCHcoz phenylacetale phenyllaclate Phenylalanine hydroxylase a mixedfunction oxidase has a tetrahyclrobiopterin cofac or 01 H2 0 am v cm Phe QCHzCHCOZ39 HOQCHzCHCOI39 Tyr H H N N N H H 1 r I OH quot K H rm N CH lseparale HN J H H enzyme 1 O OH 0 DH enahydrobiopterin dihydmhioplerin NAD NADH V H mtgm quotw on I W exped ya m Iaulomeric lorms I N an O H know the structure of lelra ar dihydmmoplenn Phenylalanine tyrosine and tryptophan hydroxylases have homologous structures phenylalanine lryptophan droxylasa hydroxylasa ldmwpdu 2mnpdn 1miwpau The active sites include an iron atom with His Glu and H20 ligands lrypto nan hydroxylase All three enzymes use tetrahydmbiopterin as a mfactor or substrate but they bind it in di orent ways 2qu mm muwpdn Tyrosine hydroxylase catalyzes the ratelim n9 step in biosynthesis of the neurotransmitter dopamine 02 02 H20 tyrosine HJNgH hydroxylase CH2 NADH NAD phenylalanine 1 Mrahydm phe hydroxylzse biopterin NHJ EH DOPA dihydro 2 decarboxylase c biopterin H2 H20 H dihydmxyphenylalanine 0H co2 H DOM DH dupamine Tryptophan hydroxylase catalyzes the rate limiting step in serotonin synthesis mephaquot sh drox mephaquot my hydroxylase Imi ngphgn H3NH 01 H20 HzNEH CH2 CH2 H NADH NAD Ho NH co2 i decalboxylase I serotonin CH2 H0 Vesicles containing dopamine fuse with the plasma membrane of the presynaptic neuron to release the neurotransmitter prasynaptic neuron dopamine in vesicles synaptic clelt gt postsynaptic receptor d t can no we quotWmquot nonconductive inquot channel ion channel Insuf cient dopamine synthesis results in Parkinson s disease Cells in a region of the brain called the substantia nigra synthesize dopamine and release it at termini that are involved in controlling motion 0 Damage to these cells can result in Parkinson s disease which is characterized by tremors and muscular rig dity 0 Treatment with dihydroxyphenylalanine DDPA is helpful 0 Drugs that inhibit dopamine receptors are effective against some of the psychi tric symptoms Dopamine must be removed to turn off signalling l I it can he repackaged in secretory Vasicles I I or diffuse to other cells where it is metabolized quotHf Dopamine is inactivated by methylation and oxidation methionine Sadenasyl dopamine W3 CHz NH MAdenosyl 029 CH3 catecholD melhyltransferase com NH MAdenosyl o c z Sadenosyl homocysteine homovaniilic acid ll 6quot coll3 OH aldehyde k7 NADH oxidase NAD monoamine all2 oxidase NAD39 NADH NH The gene for human catecholOmethyltransterase has a common singlenucleotide polymorphism that affects the stability of the protein either Vaiine NC dinitrocatechol a or methlonlne substrate analog K inhibitor SAM Sadenosylmethionine Human 108Met COMT is less stable than the 108Val variant I Pur39 39ed108Met CDMT looses its act ty rapidly at physiological temperature 0 Individuals who are homozygous for HIEMet have less issues COMT activity in thei compared to people who are homzygous for 10 al The 108Met variant of COMT is associated with increased risk for certain psychiatric disorders obsessivecompulsive disorder in men rapidly cycling bipolar disorder depressive disorder 0 aggressive or suicidal behavior in schizophrenia Particular combinations of alleles in the CUMT gene decrease expression of the 39 39 39 39 ain AIS Nackiey el al Science 31419302006 Patients with velocardiofacial syndrome VCFS lack the c gene on one copy of chromosome 2 O VCF 39 39 39 a 39 multiple contiguous genest Its in dence is 25 per 100000 births Most r Mb of mm H o CDMT is one ofthe genes that are lost I About 3 of VCFS patients develop severe neuropsychiatric conditions oh I 39 39 39 39 ui 39 tyrosine appears to help some of these patients High levels of COMT also may be associated with mental problems 5 39 u Pntta 39 mm mu c ya man Points to think about if an enzyme39s activity is not regulated closely having either too much or too little of the enzyme could be detrimental Mutations far from the active site can affect the stability of an enzyme I a r transcription of a gene or processing or stability of the mRNA Structure of the nicotinic acetylcholine receptor This slide was nm presented In me lecture It39s included here for those who would like to know more about receptors 1m neumtransmit ers view norma a the view parallel to the membrane membrane Ne Unwin J Ma Biol 346 967 2005 2b99pdb Most mammals convert aminoacid nitrogen to urea for excretion 13m em maniaAw The brok that it my gum qua 1vmlul lm l carbon chains are en down to molecules feed into the TCA cycle Some animals excrete NH or uric acid most terrestrial verteb t ra es sh amp other aquatic vertebrates birds amp reptiles i ammonium ion uric acid a i The urea cycle Hco 2 ATP 2 ADP 4 Pl 0 carbamoyl o NHA HZNlbO PO mph cilrulline ornithine NH1 0 W I E f HzNCHZCHZCHZCHCOZ HlNCNHCHchZCHZCHCOf i I V DZCCHZCHNHJ AMPPP I20 tin rlmg 3901CCHIGHNHCvNHCHZCHZCHZCHCDZ argininosuccinate arginine rim rim H2NcNHcinchI lzcn co ozcc c coz Iumarate Incorporation of ammonia into urea begins with formation of carhamoyl phosphate 3 9 NHquot HCOJ39 HzNCOIPO39 carbamoy 0 phosphate 2 ATP 2 ADP P This occurs in the mitochondrial matrix Carbamoylsphosphate w I H 39 39 Ilinntwn 1 I molecules of ATP m 9 3 nic HCOJV Ho h39o39izo39 ril acid h dride carbo phospho n v ATP ADP NHquot 2 0 carbamate HZNILIIO Argininosuccinate splits into argimne and fumarate Carbamoyl phosphate reacts with ornithine to form citrulline 30 5le Ilng DZCCHZC NHCNchHZCHZCHchCDZ argininosuccinate 9 0 my HzNtO 0 39HJNCHZCHZCHZbHCOZ ornlthlne carbamoyl 5 phosphate 9 NHJ P HzNcNHCHZCHZCHZCHCOZ39 VOZCCHCHCOI H39 cilrulline fumarale NHZ NH NENHCHZCHZCH239CHCO arginine This step also occurs in the mitochondrial matrix This reaction occurs in the cytosol Combination of citrulline with aspartate to form argininosuccinate is driven by breakdown of ATP to AMP NH ring 2 NllCHZCHZCHZCHCOZ 2302 9 in OZCCHZCHNHJ HZNCNHCHZCHZCHZCHCOZ arginine aspartate ATP WWW H D 2 AMP PP H20 argininosuncinate 9 HZNNH2 HZNCHZ CHZCHZ CH COZ ornithine 02 ll vquot 3902CCHIGHNHCNWCHZCHchchCOZ urea curs in the cytosol To continue the must return to a mitochondrion This react n oc cycle ornlthlne ts Thi A u pulll a memmane Hydrolysis of arginine releases urea and regenerates ornithine arbamoyl i i h hl HzNCOFO p p e 0 citrulline o tumquot HZNLNHZ 39DZCCHchNHJ urea arginise H10 A5 302 lisz39 llle 3902CCHCHNHCNHCHZCHZCHZCHCOZ argininosuccinate Formation of urea consumes 4 phosphate anhydride bonds The aspartate consumed in the urea cycle can be regenerated from the fumarate that is produced 2 ATP 2 ADP Pr HOD 32 carbamoyl NH phosphate oketo acids amino acids aspartate W oxaloacelate 0quot quotquot3 aminotransterase citrulline oxaloacetate urea malate dehydrogenase arginine NADH malate NAD This process also uses both eytosolic and mitochondrial enzymes Oxidation of malate in mitochondria generates ATP z e to 02 via NADH dehydmgenase 2 MP 2 ADP P generates 25 ATP NADH r 303 carbamoyl I P NA r R aspanate glutamate Ornithine citrulline mala aketoglutarate r 34 v i ornllhine cilrulline akelaglutarale glutamate um ATP aspartate AMPPPl aminoacids uketoacids argininosuncinate malate arginine Manama L mo NADH NAD and oxalaacetate can39t cross the mitochondrial inner membrane Mrm are Transport systems in the mitochondrial inner membrane exchange aspartate for glutamate and olketoglutarate for malate aspartate aketoglutarate malate glutamate H glutam me H39 aketoglutarate malate aspartate gradient because it also moves a proton across the membrane Mutations in this transporter have been linked to autism T umwmm The mitochondrial ATPADP exchanger translocase also also participate in oxidation of cytosolic NADH 2 th g zzlrzzzlty that OPEHS lo the SOIHlIDn on one Side oxaloacetate atractyiosiee an inhibitor of ATPADP exchange aspartate glutamate gtuketoglutarate malate a transmembrane u helices surrounding a cavity View normal tn the aspartate glutamate lt aketoglutarate malate membrane surlace oxaloacetate The ATPIADP translocase is the most abundant protein in the e It carries a proton into the matrix L9 57515031 2005 with each ATPADP exchange zwe pan The E coli lactose permease provides a model for other smallmolecule transporters The urea cycle is regulated in two ways The entry to the 1 39 39 39 r 39e cavity is open on 39 39de arginme 9 i0 HJNIIH acetylCoA CHZCONH ID H Naceiylglutamaie cytoplasm 2 CH CoASH Glu 0 12 transmemhra 9 me whences Caroamoyiphosphate synthetasel is completely summai inactive in the absence oi NacerylGlu hydrophmc Nace GI synthase is activat d y arginine 5 mamaL Agenetic de ciency in NacetylGlu synlhase can phosphm cause a lethal defect in the urea cycle 1 ATP 2 ADquot Pi 9 9 NHquot Hm HZNC OIPO Perivlasm closed on 0 this side Lac permease transports a proton along with a iigaiactoside across the iasma membrane G 1thiopDgaiactopyranoside J Abramscn etai Science 301251012003 1pv7puc Inherited disorders of the urea cycle can have severe effects Genetic deficiencies in some of the ureacycle enzymes can be treated pharmacologically Defective r co enzyme Effects Incidence bemm O 1 am phenylacetate ATP CokSH ATP GOASH carbamoylphosphate lethargy convulsions lt05 synthetasel early death AMP PF AMP H o san I arglnlnusuccmlc benzo ACoA 5395quot arglnlriosuccmm acidemia vomiting 15 y o phenyiaceiymm Va quotW39squot S glycine JV glutamine arginase arginemia mental lt05 CoASH o CoASH 1 H or retardation p 1 CO A lippume n 2 m griggigg v39 Approxlmate lncldence per 100000 births benzoylglyclnel quot1 People 39 quot 39 uenea uu imp LehnlngerTable la2 39 39 quot I Arginine also serves as a precursor of nitric oxide NO Autism is a neurodevelopmental genetic disorder law llHa39 arginine HZNCNHvCHZCHZCHchCOZ Repetitive or stereotyped behaviors NADPH o Incidence 1 per 1000 people possibly 1 in 200 The enzyme nitric Hydroxyarginine ADP v quot2 oxide synthase which Strong evidence for heritability bound m enzyme catalyzes both Snaps Polygenic between 5 8t 10 genes may be involved Ef39OH 39I Hi ur bou d H2NCNHCHZCHZCHZCHCO cofactors FMN FAD Singlenucleotide polymorphisms SNPs in the gene for a mitochondrial 1 2 NADPH 02 e rahydmbmplenn Caz quot quot e ulallgel V 39 U 4 i m No 1 2 quotADPquot quot10 I won39t expect you to r 0 remember these details brain Mutations in the gene impair the urea cycle and could have other H m the swam or effects on mitochondrial metabolism cltrulllne HZNCNHCHZCHZCHZCHCOZ hydraxyargmine Rama EtaluAmJ meauwm 562mm N0 acts as a shon llrec messenger in control of blood pressure blood Plottingng L Palmlerl at al EMBO 1102 506012001 The carbon chains of the common amino acids leucine provide materials that feed into the citric acid cycle lysine phenylalanine tryptophan acetoacetylcoA isocitrate QIU amate citrate uketoglutarate succinylCOA nxaloacetate succinate isoleucine methionine threonlne valine asparagine aspartate Amino acids can be clas as glucogenic or ketoge ketogenic Some fall in both groups iso39eucme I E isocitrate leucine threonine tryptophan 1 uketoglutarate isoleucine methionine succinyI CoA succinate malate fumarate glucogenic oxaloacetate l asparag ne aspartate tryptnphan Humans can synthesize 10 of the 20 common amino acids Essential and Nonessential Amino Acids umans Amino acids that an for H synthESize in adequate Essential Nonessential ounts under a given ne lne Histl lne Asparaglne lsoleucine Aspartate Y Leuc39ne Cysteine quotonessenual39 Lysine Glutamate Mo bacteria and Methionine Glutamine an s can Phenylalanine ycine synthesize aquot 2 Threonine Proline ammo ac39ds39 ryptophan Serlne Tyrosine 39Arg is essential in infants and growing children but not in adults Building blocks for synthesis of nonessential amino acids in humans come from glycolysis and the ci 39c acid cycle glucose glycine 3phosphoglycerate s serine cysteine pyruvate v alanine citrate proline oxaloacetate aketoglutarate gt glutamate gt glutamine Dana s K arginine asparagine Sarina is formed via 3phosphoglycerale 502 c H OH co HzOlt H 3phosphogiycerate MAD NADH 3phasphchydraxypyruvale Glu gt akG 9 2 H NCH col H N H HzOH 2 HZO lphosphcseiine serine P quot20 The first product of CO2 xation in green algae is 3phosphoglycerate M Calvin a A Benson exposed green algae to Hcoz for s on periods onime in the presence of ligh ide pape chroma o alga boiling methanol light quotc appeared rst in the carboxyl carbon or I701 3phosphoglycemte quotbequot I CHzo t ey separa an ntified the labeled products by r t grapny 3phosphoglycerate is formed from ribulose15hisphosphate In another experiment Calvin a cowarkers lirelabeled all the cellular urn in me riarlr III III whose concentrations decreased when they turned on a light Lero CH 0 If H20 2 H39 HIJOH r AA Maori 39 rihulose hlsphosphate H39C39OH carboxylaseloxygenase Imz H2o rublsco HcDH ribulose15bisphusphate CHICG 3phosphoglycerate 2 molecules Ribulose1Sbisphosphate can be regenerated from 3phosphoglycerate if ATP and NADPH are provided 39202 ATP ADP Dco NADPH NADP CH0 I H40H 5 4 HclOH 3 lt rug0H CHI 06 v cuo Pl 139 phosphate CH D Em Em i8 E H I0H I M E Halli0H many steps H OH rilniloee 15 Hz bisphosphate CHID glyceraldehydea phosphate Stoichiometry of CO assimilation in the e Calvin cycl E I E n l E ribulose 15bisphosphate E E l 5 eraldehyde 3phosphate F J 39 m glyceraldehyde 3phosphate Regeneration of pentose phosphates by the Calvin cycle CHZOH Inlon co xylulose SP 5H0 erythrose 4P CH0 glyceraldehyde 3P xylulose SP sedaheptulose7P Four enzymes of the Calvin cycle are activated by reduction in the light The a mall HS SH 7 L 5 5 EHZ me lnaEllVe from PS I g Fructose16bisphcsphatase is activated by increases of pH and Mgz in the stroma g 150 39 l H a 0 When chloroplasts are E p Illumlnated the p In E the str ma rises from 3 100 7 to 8 and Mgi39 3 increases lrom 2 mM o 5 mM 5 w 50 0 II I a an o o 5 15 20 R U I u Isco a so Is Mach quot1quotquot activated in the light but by still another mechanism Lannmgev Fig 2045 The carboxylase reaction catalyzed by rubisco proceeds through enzymebound 6carbon intermediates Carbamoylation of Lys 201 at the active site of rubisca is inhibited by bound ribulose15bisphosphate o E quot239 s quot2 g fHZo Anomer enzyme rubisco 1 535343 my 5 CO Hocco 39 activase catalyzes ATP 39 8 I z Z39Cal bOXY39339k939039 dependent removal of cHD qpia mlll aquote quot f39o i IZDH o arabinitoI15 ribulase bisphosphatet 10 5 he iigm by mimicquot HItOH H g Hli OH 490quot 39SPquot 5 quot3 who of rubisco activase ribulose 15 i CH239 i c CHz0 OH39 bisphosphata HCOH rublsco rihuloselt1 39 CH O mm Ho nonenzymatic bisphnsphale I 2 carbamoylation CHr C ATP ADP P co I I I l coz 2 H cHzO Hoccoz Hocco HCOH NH3 NH NH Hmlzi quot I mm 2 0 oc quotz39 f 39 m NHCHCOm NHCHCO NHCHCo 3phosphnglycerate 3phosphdglycerate inactive inactive active A carbamoylated lysine side chain forms part of the binding site for M9 and 45Z in rubisco Ruhisco also catalyzes an oxygenase reaction that generates 2phosphoglycolate ribulosedj bisphosphate phosph e 2phosphoglycalate oco lo Zcarhoxyarablnitol blsphosphate an inhibitor 10 is an analog of e n rmal product Its carboxyl group probably occupies the col binding site 3phosphoglycerate curo Phosphoglycolate is oxidized wastefully 39 es in mitochondria and peremsom 02 CH O IV A comma an multiple steps NoATPI phosphoglycolate I ni 39 39 39 39 39 39 NADPH are available for synlhesis 0f ribulosa bisphosphatel nfthe coz they mt Some plants have mechanisms to minimize photorespiration Q1Ho gt HCOJ ItH PEP Pl 0 oxaloacetate mescphyll cell E mm CH2cco 02 Cquot 410 phosphae NADPH dikinasa A AMP ppl gt quotADP 0 ATP P 9quot II CHcco 39 OICCHZCHCO A l l Typical architectures M C and 6 plan s mesophyll cells C Plant bundle sheath C plant can bundle sheath cells have many chloroplasts bundle sheath cells have few or rm chlarap asts wwwcculeiparklnsmphowppl Plasmodesmata connect the bundlesheath and mesophyll cells Plasmodesmata are channels that allow Lehnlnael Flg 2023 cell wall 2321 The plasma membrane is MESOPhY 59quot continuous between the two cells Nothing in biology makes sense except in the light of evolution T Dobzhansky Biochemistry 441 Lecture 2 Ted Young January 9 2008 The utility of complementarity Denaturation and renaturation of nucleic acids Denaturation of nucleic acids ds DNA The forces that hold the two strands of DNA together are all weak forces and therefore the two strands can be easily separated Denatured random coil Common denaturing agents in the lab Heat high pH and strong H bonding agents such as urea and formamide Why not water Why not low pH 7 To function in Viyo as a template for DNA and RNA synthesis the two strands must be separated using enzymes known as DNA helicases Denaturation of nucleic acids ds DNA The denaturation or meltlng of double stranded DNA occurs over a very narrow temperature range 1nd1cat1ng that 1t IS a hlghly cooperatlve process The temperature at the nndpolnt of the meltlng curve IS known as 1ts rneltlng temperature or Tm 14 Relative absorbance at 260 nm H E M w Equot H 10 lt Transition breadth gtl 100 50 Percent hyperchromicity Energetics of denaturation AG 2 AH TAS helix coil One molecule two molecules Few conformations For the helix to be stable AG must be gt0 AS SCO11 S helix gt 0 therefore TAS is negative Therefore for the helix to be stable AH must be larger in a positive sense than TAS is negative AH gt TAS AH is positive because there are strong interactions between basesboth Hbonds and base stacking due to van der Waals interactions between the bases These must be stronger than the repulsive forces of the negatively charged phosphates which are partially neutralized by interaction with Na K and positively charged Lys and Arg of the histone proteins many conformations Energetics of denaturationcontinued As the Temperature increases AG approaches 0 At the Tm AG 2 0 and Tm APUAS AH depends on base sequence and composition Whereas AS does not therefore helices With higher GC content 3 bp versus 2 for AT pairs and more stacking of GC pairs Will have a higher melting temperature Basepairing in nucleic acids CH3 I Majorgroove Crlek base pairs Note that for both GC and AT bp the hydrogen bond donors and acceptors shown are the only ones possible if the positions of the Cl atoms maintain the geometry of the B heliX Other pairing can occur in unusual DNA structures for liMinor 2 example at the ends of Chromosomes Hoogsteen base paired G quartets are found Basepairing in nucleic acidsevidence for pairing in solution Infrared spectra of bases NH region of the spectrum W 13 G 00008M W A Relative absorbance kL l A 00008M G C 00016M Calculated sum A G A 00016M Observed Calculated sum 3500 3400 3300 Wave number cm 1 Only for the pair GC does the IR spectrum change implying that the N H bonds are altered this change is seen only for standard Watson Crick base pairs G C and A T not GT AC TC or GA 10 Basestacking plays an important role in stabilizing doublestranded DNA Studies of the osmotic pressure 7 of solutions of free bases demonstrates that they aggregate 7 RTm Where Rgas constant Ttemp mmolal concentration a measure of the number of molecules in solution For substances that aggregate 7c RTm Where osmotic coefficient which has a value between 0 and I in other words the effective number of molecules in solution is reduced by aggregation and this reduces the osmolarity Evidence for base stacking in solution 10 The reduction in I is not due to H bonding between 09 bases since N6N6 methyl Adenosine adenine can t H bond and shows the greatest reduction in I 0 oo 2390 Methyladenosine 2 Deoxyadenosine smotic coefficient 4 o l O 0 07 I N6Methyladenosine 0 01 I I 6Methyl239deoxyadenosin 04 I I i N N 6Dimethyladenosine I I I 01 02 03 m 12 Tm can be used to characterize DNA from different organisms Melting temperature is affected by GC content 10n1c strength and solutes that affect hyerthbrc and H bondrn 1nteract10ns In enera a ents that isrupt H 1nds or base stackrn increase ecrease the Trn LOW ionic strength dashed curve increasesdecreases it 100 80 015M NaCl 0015M Na citrate 0015 M NaCl 00015M Na Citrate w 522 Trn continued Trn does not depend on size of the DNA after a certain size about 30 bp is reached For short oligonucleotides Tm150AT 350 GC in Ocentrigrade ATGCnumber of bp For a large DNA of 50GC the Trn 850 so obviously the relation fails after a certain size For a DNA oligonucleotide containing only Gs and Cs What is the largest size for which this relationship is valid Structure of denatured DNA Rapidly cooling of denatured DNA leads to imperfectly base paired single strands of DNA The figure illustrates intra strand base pairing that has normal WC complementarity and orientation Questions to consider About how much base pairing would occur when a DNA solution is rapidly cooled Would all denatured DNA molecules form the same structure Would both strands of a double heliX form the same structure Would single strands of DNA and RNA of the same sequence form the same structure Intramolecular aggregation s 4 Intermolecular aggregation 15 Intercalating dyes can distinguish doublestranded from singlestranded DNA in cells ssDNA Red luminescence Ac dim Ovirlgu w Jillnu dsDNA v Green uorescence is Acridine orange binds to DNA and RNA in the cell RNA in cytoplasm DNA in nucleus Why are these bodies the nucleolieorange This pan ofme slide wax llealed wilh NaOH How can you explam the difference Whm would you have see If hem mmcr than NaOH treatment had been used 17 DNADNA and DNARNA hybridization Singlestranded nucleic acid molecules that are completely or even partially complementary will react with one another Via hydrogenbonding to form stable doublestranded molecules radioactive heat D A RNA Stab 39 EM of partially denatured DNA Annealing or hybridization of two related but not completely complementary singlestranded DNA or RNA molecules would Produce a similar picture the locationand size of the single Stranded regions would identify the location and extent of sequence divergence Annealing of nucleic acids is important in most eXperimental manipulations in the laboratory DNA sequencingDNA synthesis to make probes Polymerase chain reaction PCR oligonucleotide mutagenesis creating recombinant DNA molecules detectingmeasuring DNNRNA isolated from cells determining relatedness between species formerly Separation and reannealing of DNA strands is important in vivo Every process we talk about with one exception involves separation of DNA strands and rewinding of the helix Strand separation involves putting energy into the system usually in the form of ATP hydrolysis using enzymes and proteins that are involved in unwinding of the DNA 21 2 Phospholipids triacylglycerols and complex lipids Membranes contain a variety of glycerophospholipids and sphingolipids if cHO VWWVVV stearic acid 0180 9 CHDC 0 390 CHIDTOCHZCHZmc zl 0 glycerol phasphoc39noline o O CHICHZ N CHS sphingomyelin nleic acid C1B1A9 1slearoyl nleoyl phosphatidymholine uzher common glycemphcspha imds e pnuspnauaylemanmamm phosphatidylserine phusphatidylglycero phasphatidylinosilol snmnquupsas with carbohydrate mu 5 serve as recognitio lemurs on cell Surfaces Lipids in rat hepatocyte liver cell membranes liar hepararyre membrane type Percent membrane lipid The fluidity of a phospholipid bilayer increases at a characteristic temperature Tm where the side chains become more disordered short side chains melt at lower temperatures Most natural unsaturated fatty acids rrrs h39 pack latly acid si l hat have de ha39 do well with phospholipids t Temperature M it coz linulenil acid Unsaturated fatty acids and shortchain fatty acids make natural fats more fluid Fatty acids 0 of total C16 amp C18 saturated C16 St C18 unsaturated 4 to C14 saturated liq Natural fats at 25 39 e oil Butter Beeffm uid soft solid hard solid Cells adjust their phospholipid composition so that Tm typically is slightly below the growth temperature Fig livmg outdoors in winter t cool a warm Ratio of u nsatu rated temperatures Culture Temperature C AG Mar amp JL lngraham 1530 EA 1260 1962 Animals snthesize oleic acid C181 from stearoleoA by an enzyme system that uses molecular 02 as an oxidant WED54 stearoyICoAC18o 02 NADPH coscm 2H2 NADP olecleoA ClB1A This 39 39 39 electrons for me reduction of O2 to water which requires 4 electrons ozue uir 2H1 A electrons from NADPH to he oxidase palmitoleate 0161A5 is synthesized similarly from palmitate Animals can t synthesize unsaturated fatty acids with two or three double bonds linoleate 1321AM linolenate133A9 12 151 Linoleate and linolenate are required in the diet They re essential fatty acids But once ingested they can be converted into 0 er 4 Mquot Triacylglycerols are synthesized from glycerolSphosphate nd fatty acyl CoA thiueslers o D 0 II E RCSCoA CHZ CR R CSCoA D Imrmm HOKIDH I R COCH Lquot CHzOPO39 cHzoPo CaASH I CoASH b glycerol1b phosphaudic phosphate acid H20 3 szocR CH P CHEOH triacylglyceml diacylglycerol Adipose tissue can generate glycerolsphosphate from glucose or by glyceroneogenesis ATP ADP so GDP f col P I an co cu2 0 50 c0 I u I COPD CH3 H2 phosphoenolpyavate L1H 5 Draft 902 carboxykinas Z PYWVE E V oxamce m phosphoenolpyruvate ATP ADP 1 AL gt NAD gtNADH P CHZDH quotADquot AD Izuon glyceraldehyde IFO 1quot quotofquot Iquot 3 hosphate cuzoPo INTOI39M 039 o glycerolSphosphale dihydroxyacetonephosphale Glyceroneogenesis and gluconeogenesis are regulated reciprocally by changes in the amou o FEPcarboxykinase in liver and adipose tissue ad ose liver tlssue 421 fa y acids triacylglycaml j K Kx f 39 pymvau thiazolidinedione dru s used to lreattypeZ diabetes choline Mammals synthesize phosphatidylcholine and phosphatidylethano amine from cytidine nucleotide derivatives of the bases ATP rlm2 H MD GDPcholine a ct PP H o 0HNCHZCHZOH 04 5 5 i CH2N39CHzCH239 CH3NCHZCHzDFOI mmquotz 5 o39 o phasphuchallno g H H 9 IZHr 4 gtCMP R COCH v diacylglyceml 4minquot I o CHocR Q I R OCH n phosphatldylchaline JHz oquotquotM3116HerCNm D Fhosphatidylinositol and some other glycerophospholipids are formed from CDPdiacylglycerol NH1 0 0 AL I ll o CHrDCR 11 PP o szOCR E Hquot II R39IOiH D E z RquotC0 fH o o 04 N H 39cnrolio CHrolLDroCHZ 39 o o o phosphatidic acid CDPdiacylglyceml OH DH 0 0H 0H II D liHiOCR H0 ll H R C0ltfH 0 OH OH OH II b CHIOPD H b inosilol gtCMP phosphalidylinositol 0quot H Phosphatidylinositol participates in intracellular signaling 0 CHZoER I D phospnalldylinositol CHOJLRr phosphatldylinosiml D 415bxsphosphate i l CHZ Oiio 0H 0H CHzolfo 0H OH gt o 0 H10 H H rrnone sensltlve phosphollpase in plasma membrane r diacylglyceral inosiloI1 4 trisphosphate v activation of protein kinase C u release 0 ntraceilulal Caquot v v regulation of other enzymes regulation of other enzymes Eicosanoicls are 20carbon lipids that act locally to stimulate a variety of processes Frostaglandins co CH act through oAMP to stimulate contraction of on oath musoie eiieet biooo iiow eievete body 0quot temperature or cause iniarnation 8 pain Prostagiandin E co N Thromboxanes CH 0 produced by piateies act in iormation oioiood quot oiots i reduction of blood ow at the site oi a ciot Thrambnxane AZ 0 CU Leukomenes 5 induce contraction if muscle lining airways to the iungs Leuko lene A I won39t expect you to remember these structures Mammalian cells synthesize phosphclipids containing arachidonic acid eicosanoi s from arachidomc and phospholipase A1 0 iysophosphclipid Arachidonic acid is reieased by stimulus quot 60239 arachidanaia breakdown ofphospholipids in m 2 response to ell age or hormonal stimuli z o cyclooxygenase z aspirin 1 ibuprofin coquot ammais have two isozymes of o nase cox1 s cox2 The OOH pros aglam n 52 9 produced by cox1 cyclooxygenase participate In quothousekeepingquot functions suc as secretion of gastric m 39 those produced by cox2 play roles in o co innarnation I 1 E Aspirin ibuprnfin and acetaminophen 0H prostagiandin H1 nonsteroidai antiin amatcry drugs NS IDS pieck prostagiandin synthesis by inhibiting cyclooxygenase other aimsamms Phospholipase A2 and cyclooxygenase operate on substrates in the plasma membrane View parallel to membrane View normal to membrane r quotJyquotsl y I I ma phospholip J l membra idhilayer I I I I I I I I I I I I I w notelhelunnelsleadlngfrcmme y I a I I I y l a 0 M I membranetotheamivesiles The cyclooxygenaseZ dimer with bound Evumb arachidonate viewed from two perspectives Cyclooxygenase 1 is structurally very similar to cyclooxygenase 2 View parallelto membrane view normal to membrane e y rllJJ IlJIl I membrane phospholipid ll w lll wMIm the heme is at the peroxidase slte cyclooxygenase1 dimer viewed from two perspectives 1qupdr Nonsteroidal ant nflamatory drugs NSAIDs have been used widely to treat osteoarthritis and rheumatoid arthritis Aspirin taken regularly in small amounts decreases the risk of atherosclerosis Aspirin and other drugs that inhibit both COX1 S COX2 89 ibupro n 3 gastric mucin causing stomach irritation Drugs that speci cally inhibit COX 2 cause less stomach irritation a nu and valdecoxib Bextra were found to increase the risk of heart attacks and stroke Similar effects were found with naproxin Naprosin Aleve which inhibits both COX1 3 CO 3 mm m Ribonucleotide reductase reduces ADP GDP GDP 8 UDP to the deoxyribonucleotides X ribonucleotide Twocysteinesin 5 reductase the enzyme serve as the redumam NADP39 NADPH Ribonuc eulide hich in me cytcsm Each of the catalytic sites in ribonucleotide reductase has a binuclear Fe center and a stable tyrosyl radical S has two types The enzym of subunits R1 and R2 regulatory sites The catalytic ites are at the R1 R2 interfaces lA8pdb O by Js two Fe atoms with His Glu a Asp ligands Ribonucleotide reductase generates freeradical intermediates 0 0CH10 NDP H H 0H 0H e tyrosine Th radical generates another radical X39 Close to the substrate 0 04 H10 H H DHH Steps 1 St 3 H atom H transfer Step 2 hydride ion H 39 transfer or two 1e 39 reactions and H dNDP Must eleclru ransfer reactions 039 NADH Involve hydrlde lon lransfe D 5 lation 39 39 reduct quot t 39 site balances the rates of formation of the different deoxyribonucleotides dTTP inhibits allosleric reduction of GDP 8 EHEC WS DP and aciivates ATP dATP reduction of GDP dGTP di39rP ATP activates reduction of GDP 8 UDP su bslrate speci city site activity regulation ATP dATP site dATP inhibits and catalytic ADP GDP 539 UDP GDP substrates At the activityregulation site dATP inhibits RNR and AT increases Vmax for the reactions of all the ribonucleotides substrat specificity site activity regulation site ATP 39 dADP gt dATP sataiyus site the relativ eaff site ATP dATP d39l39rP and dGTP tune 39es of RNR tor CDP UDP GDP and ADP ATP CDP dCDP 39gt dCTP UDP v GDPagt dGDP gt dGTP vv 9gt dUDP dTI39P Two sets of redox proteins carry electrons from NADPH to ribonucleotide reductase RNR NDP x RNR SH SH glutaredoxin I5 S glutarednxin reduclase 4 glutathione 2 GSH GSSG glutathione reduclase NADP NADPH gtltI gtlt gtlt dN DP x S S SH SH X X RNR SH IS SH 5 lhioradoxin 739 SH SH thioredoxin reductase FAD NADPH NADP CHZSH H3NFHCHZCHzCONHbHCONHCHz00 CO 39 Z Glutathione yGluCys Gly is the main redox buffer in the cytosol of most eukaryotic cells Structures of reduced and oxidized thioredoxin in solution C32 5 I reduced 1trwpdb oxidized 1trstpdb Thioredoxin and glutathione also play important roles in detoxifying reactive oxygen species Schistosome parasites which Ehhgggcwoq zpdse E12 33 are responsible for more than 280000 deaths annually have Lillig 5 A Homgrenv only a single multifunctional Am39c x39d39 Rad S39gnaL 939 25 enzyme for detoxifying reactive won 0 species inhibitors of this e a promising way to treat schistosomiasis AH Sayed et al Nature Medicine 14 407 2008 Thymidylate dTMP can be synthesized from either CDP or U0 5quot ATP ADP hf CH CDP dCDPAL dCTP M H 2 0 N ribonucleo de nucleoslde dCT diphosphate deaminase reduclase kinase gtNH3 I UDP dUDP 7T dUTP H20 HIERBIquot ATP ADP K gt PP H I 04 N V 5 0 dUMP Wmmhvlene 0H 0H 9 TH A A hymidylate H c synlhase Hydrolysis of dUTP looks dTM H wasteful Why don39t cells use 0 N dUTP To make d H39P directl k y I ATP dTrP In the process of methylating dUMP thymidylate symhasa I izes N5N quotmethylenetetrahydrofolate to dihydrofolate E lgtlN ltgt 1MHGlun 78dihydrofolale H WNWmethylene leuahydrofalale o 4 quot legellelale using NADPH as the reductant H H HZN N dihydmfolale 2quot N Y I H reduclase Y H N N gt quot N H OH HN R 0quot N etiahydroiolale 75dihydroiulale NADPH H NADF Serine serine hydroxymethyl ransferase 1pr Glycine HZNYN 5N melhyiene N tetrahydrofalale H N Enzymes of nucleotide metholrexate biosynthesis provide targets N N o or cancer chemotherapy NH CH Gil NH mu 3 o i Analog of DHF Inhibits 139 mil F dihydrofolate reductase CH thymidylate synthase D cx N H Hu c cF inviva o VAN 39 Qquot gt gt 39D OCHID 0 Analog Of dUMP Inhibits H S uomuracil FdUMF co 3235mm H3NbH Analog of Gln inhibits glutamine DAdiazoacetyl quot amidotransferases snaps 1 ii 4 of Lserine I 2 purine biosynthesis CTP synthetase D amp carbamoylphasphaie synthetase ii Primates oxidize purines to uric acid for excretion l quot1 l P N C N N WK 0quot W l H W s 0H Hc NCN HZN RN KN Ho KN KN H ribose libese urlc acld adenosine guanosine 0quot C N 4 F C 0H Birds reptiles and insects also excrete uric acid Hoc NCN Fish and most terrestrial mammals oxidize uric H acid further before excretion N uric acid has several W E Fo tautomeric forms o kN K a Formation of uric acid from purines hypoxanthine adenosine inosine N o o I 1 N 0 NH 3 P ribose1P E N N 91 C S HN c C HN c Hc 3 Cquot d Hc39 l39 quot H6 139 H a enoslne N I deaminase N f N u ribase ribcse H I 02 H20 6 N c N lt HN c HN c C H10z xanthlne H N quot H gt gt 5 393 H oxidase 1 NC N Ho N N guanosine ribose xanthlne Mum 02 H20 39 A E Xanthine oxidase contains FAD a xan hine H102 HN fN two FeS centers oxid I ll fDH efre purines Ho C N u re th not the nucleosides or nucleotides Mutations in adenosine deaminase cau Severe Combined Immunode ciency Disease deoxyI NH 20 NH 0 deoxyinosine adenosme I A L N v L4 MK 6 uric HR 393 3quot ade asine Hg J H acid N Ili data ase N N deoxyribese deoxyribose u ir I levels u ir39 quot 39 39 39 dAITP of other deoxyribonucleotides and cuts off DNA synthesis Lymphocytes 5 Pan W F 5m my See Garrett amp Grisham pp 398400 V ribonucleotide reduclase ribonucleotides gt deoxyribnnucleotides gt DNA Deposition of sodium urate crystals in tissues causes gout iqu Crystals ful in amatian of sodium urate form in the toes kidney and other tissues causing pain hypoxanlhine xanihine uric acid 9 g g c xanthine X8 quot19 HN C N Maia HN c N quot359 HN c N ml E CH 5 CH I g 2 0H W N V Ho N N V HD KN N H 02H10 H201 H oHo H20 H I i i c E Gout can be treated with c N Na HM aliapurinol an an lo of if E co H NcN hypmfanthine that inhibits O KNRN H xanlhine axidase H sodium urate allopurinal Salvage pathways regenerate nucleotides from free purine and pyrimidine bases NH adenine nu phosphoribosyl 3 12quot NArN transferase RCk H CH quot H HC NCN 7T HC MCNC H V am a 9 9 PH FDmm o mg of o 0H 0H W 0 0 AMP OH OH 5Aphnsphorihosyl l Asimilaren me h oxanthine uanine pyrophosphatelPRPP ZY VP 9 phosphoribosyl transferase wo k hypoxanthine and guan r s an ine Mutations in this enzyme lead to LesohNyhan syn rome Lesch Nyhan syndrome l n l u seCere neurological defects mental retardation selfmutilation cerebral palsy elevated uric acid gout LeschNyhan Syndrome can be diagnosed prenatally normal Fetal broblasts obtained by amniocentesis are u cultured in the presence of 3Hhypoxantl tlne r e 39 It which precedes to AMP and GMF and is incorporated into DNA LeschNyhan Drug design cycle using protein structural information Experimental Target Protein Computational Functional Genomlcs Idenli calion Functional Genomlcs Target Protein Overaxpresslon so Structures ul Target Proteins t ProteinDrug Complexes t on t V V V 333 53 Data Base of ProteInDrug mama studies Chemlcal lnlulllon Complexes onemloal Synthesis oomolnaton al onemlatry x ay NM electron croscopy For examples see wwwl1rnscwashingtoneduNVimHolsummary Clinical trials of drugs are conducted in four phases Phase Size Goals 2080 Evaluate safety determine safe dosage range identify side effects II 100300 Evaluate effectiveness further evaluate safety quotI 10003000 Con rm effectiveness monitor side effects compare to other treatments Iv pas marketing Collect additional information on the drug39s risks bene ts 8 optimal use Doubleblinded tests with placebo controls re required in Phase I receive testdrug receive placebo or other drug with proven effects large pool of potential mi gimme key to assignments kept secret BIOCHEMISTRY 441 Winter 2009 1 Biosynthesis of fatty acids 2 Triacylglycerols phospholipids amp complex lipids 3 Cholesterol Br lipoproteins 4 Photosynthesis antennas Sr reaction centers 5 Photosynthesi electron transfer Kr photophosphorylation 6 Photosynthesis carbon xation by C3 and C4 pathways 7 Amino acid meta olism transamination and NH3 transport 8 Urea cycle amino acid catabolism amp biosynthesis 9 Aromatic amino acids Br neurotransmitters 10 Onecarbon metabolism 11 Biosynthesis of pyrimidines amp purines 12 Deoxyribonuoleotide biosynthesis Sr nucleotide catabolism DNA and RNA structure Automated OnIine Video Screencasting is used in this course Screencast recordings of the classes are located at httpwwwcsswashingtoneducourseBIOC441A To watch a Screenoast right click the Screencast link Adobe Flash Player is required Visit the Adobe Flash Player Download Center if Flash is not installed on your computer I3939 It It lnha Fatty acids have extended hydrocarbon chains 1 16 AAAW902 palmitic acidc16a 1 quotAvwvwvcozn stearic acid c1so 9 1 in 50 NJWVV 9 1 meow oleic acid C1B1A9 15 Most natural fatty acids have an even number of carbons Most natural unsaturated fatty acids have sis double bonds paimitoieic acid 0161A9 Fatty acids or glycerophospholipids sphingalipids triacylglycerols o EHED oR 0 H0131 CH CH CHZR o HZOER R EOICH Rquotl39NHICH D R JLOICH o CHZOIITDCHZCHzNlcm cup ocuzcnrmcng cuzolLR o o v I quot Sphingoiipids on cell surfaces Triacyiglycerois are l are sites otceii recognition stored as energy l l e es inositoi phosphoii pids participate in intraceiiuiar signa ing lti 39l l structural elements of biological hranes cholesterol esters in iipoproteins and are attached covalently to some protein capillary Triacylglycerols are stored as energy reserves in adipose tissue and other tissues i l Bun cytosol lipid droplet Cross section of four adipocytes from a guinea pig Lipid droplets consisting mainly of triacylglycerols fill most of the volume of the cells Fatty acids are synthesized from acetylCoA in adipose tissue amp the liver ATP NADPH CO gt gt gt O Wa39s39 Hsc palmitic acid C1620 palmitoleoA 0 gtSCOA H3 stearic acid c1820 stearoleoA 9 0 SCoA oleic acid C1B1 A9l oleoleoA The labeling patterns suggest that the fatty acid chain forms by successive addition of twocarbon units MalonleoA serves as the donor of twocarbon units malonleoA P gCHZ JSCOA 0 mmquot 9 ATP ADP ll CHaESCOA I c c c HN NH 9 Pi HM Wow HN NH 5 s s s o NH Mlnlr A39 mm by a 9M covalently to a Lys residue of the enzyme ln bacteria the three domains are in r they are Pyruvate carboxylase a similar multifunctional enzyme has four domains with different functions The active form of pyruvate carboxylase is a tetramer biotin carboxylase BC blue carhoxyltranslerase CT yellow biotin carhoxyl carrier protein red allosteric regulatory domain green ATP v Zn one monomer M St Maurice et al Is oumned Science 317 1076 2007 2ql7pdb The biotin carboxylcarrier protein moves carboxybiotin between s39tes on different monomers How could you test this Hint make a mutant with an inactive BC site and another mutant with an inactive CT site The fatty acid chain is synthesized attached to a protein acylcarrier protein ACP Malonyl and acetyl groups are transferred from CoA to a thiol sulfur atom of ACP O malonylCoA 39OZCCHzltSCOA 0 aoetylCoA cu6sCoA ACP of Bacillus sublilis 1f80pdb In animals ACP is a domain of a large protein HS gt a H54 Bacteria have H5 1 HSCoA The condensation reaction malonyIACP 3ketoacylACP O P 0W SH 32 P olecurbs malonylACP malonyllacetyl transferase In animals39 both reactions 0 CHE36 acetyl ACP The acetyl group flrst moves tram ACP Release at bicarbonate is o a m 39 i 39 A ru39m r 39 h 4 give a 3AketoacylACP condensation The functional thiol group of ACP is in a 4 phosphopantetheine group linked to a Ser residue of the protei CH 039 o I0 quotI Hs F OSer VKN g H H a P if CHaJiS OCCHz centers in bacteria it39s a separate small protein ketoacyl synthase Hco H Reduction by NADPH H followed by dehydration o o and a second re uction CH33939CHZ39 ph05phopanletheine generates butyrylACF NHz 5 u ketoacyl NADPH N c d ct CoA SH ml 2 gt2 399 quot 59 NADP 0 ID quot339 H sN N CHICH2sz a UP O CH HsNI N E g ID and lreductase Ii 3 H n on NADP y mfgskirt 4v v 0 0H dehydrase 0H the thiol group otheriZyme 7x Lolz quotADPquot Pantetheine is Vitamin as H o I 393 4 lt CHJ39C39F 39 HOH H To continue the cycle the fatty acid chain must move back to the ketoacyl synthase 0 I i CHz39 5 OCCH2 O 393 cmCHZCHZ s 5 if u cureonio H10 P Bakaloacyl NADPH CHSICHECHT redumse NADF Am enuyl reductase T E CH CCH C dehydrasa z 2 NADPH CH 9 0H 3 I HOH The second turn of the cycle generates hexanoylACF 13 o OCCH2HI CH CH CH E pkctoacylsynthasc coZ 139 Z 2 39 H 9 3 CHJCHZCHzCvcl lzC P ketoacyl NADPH d l CHJCHZCHz CHZCHz39J S 399 quot N gtNADP NADW enoylreductase dehydrasa ill 139 CHCHchzcCH c H NADPH H 0 0 CH an lt39 393 4 lt 2 E HOH The cycle continues until the fatty acid chain reaches 16 carbons Thioesterase hydrolyzes palmiloylACP cquot i releasing palmitale 0160 0CCHZ 0140 O cnacnzmcurlbs 5 9 if m mim msa CHICH1 CHZCCHzC H30 NADPH cmcngncmCHZCHZc gt NADPquot ketoacyl synthase SH 2160 H O NADP CH CH CH IC CH quotC NADPH v I 139 2quot 22 239 39 CH6H2HCHzccb lt lt h HOH Chainlength speci city of the substrateloading chainelongation and chaintermination ac it39 of mammalian fattyacid synthase 12 A E E E a 3 E E E 0 E E g 2 a 1 l m V g V a gt c In E a 3 gg gt5 E E 1 f g 3 gtc a g m 9 2 E I5 2 a I 39 a 3 a 2 a E g I E 0 U 0 u o u u S Smith et al Frog Lipid Res 42 289 2003 The enzyme protein during evolut Animals one multifunctional protein H co OH H N H 00239 mu co 3 H Ophosphopantetheine s of fatty acid synthesis have fused into a single ion art90239 0H 0phospho pantetheina Architecture of the mammalian fatty acid synthase The active form of the enzyme is a dime The ACP 8t TE domains are not resolved in the crystal structure probably because very they are mo ile The white and blue spheres Indicate the active sites Hollow spheres in antheteine showing the domain colors represent the length of phosph how closely ACP must approach each site during Mater el al Scrence 311 1258 2006 ch2pdb 3R 6 catalytic cycle Yeast fatty acid synthase contains two proteins each of which has 4 different types of catalytic sit es Six copies of each points for the ACP domains Lomakin etal12007 Cell 129 319332 Jenni el al 2007 nce 316 254 Differences between fatty acid synthesis and oxidation oxidation liloi mitochondrion o H WOO5420A F NAD NADH o AJK COSCaA NCO5420A synthesis cytosol Fatty acids with longer chains C180 amp 0200 are synthesized from palmitoleoA 8i malonleoA by an elongation mechanism COSCDA palmimleoA C16 390209 mSCOA These reactions occur caAsH 02 in mitochondria amp the 39 NADFH K NADF Dilfarent enzymes ar H o in place of ACF but the 1 NADFH reactions are otherwise ionnally the same as in b NADF synthesis of palmltate 0SCoA stearoyICoA imam Citrate carries Zcarbon units from mitochondria to the cytosol citrate Mitrochondrion syntnase GOASH acetylCoA Wm 3 093900139 Hobcoz I 2 H oxaloacetate coz co 2 citrate Citrate lyase uses ATP to drive the breakdown ai citrate to acetleoA a oxaloacetate in the cytosol Integration of fatty acid synthesis with carbohydrate metabolism Cytosol Mitochondrlon pyruvate A39s ATP CoASH f ADP r citrate 3 ATP lc A y i new 0 ADP acetleoA P oxaloacetate oxaloacetate malate NADH NADH aehyum genase NAD 20 gt NAD 9HOH malate malate CH2 oz NADF39 mall my be NADFH co pyruvale pyruvate AcetleoA carboxylase biotin carboxylaseltranscarboxylase is the main control point for fatty acid synthesis in animals the enzyme is regulated by both allosteric effects and phosphorylation insulin stimulates the phosphorylated dephosphorylatian enzyme is inactive actlvallon acety M lt A CAMPdependant protein kinase acetleoA malonleoA glucagon epinephrine and adiponectin stimulate phosphorylation inactivation palmitoyICoA 1 malanleoA inhibits camitineaeyltransterase I blocking transport ofpalmitoyl 39 I UAIUGHUII The active unphosphorylated sympathetic mmquot 9339 mmws of the brain form of acetleoA carboxylase system forms long filaments neuronal signals Increase cataholism a thermogenesis express gene for Increase uncouplin protein blood 9 ad tin pressure 3 heart rate Decrease fatty acid synthesis nd increase catabnlism phosphorylzte acetleaA carhoxylzse In an iiv An imbalance between energy input and output can lead to obesity Defects in leptin or its receptor can cause obesity adipose weight weight 67 g 35 g 65 of the 2mm u e V ea 35 ar 39 m typeli diabetes and cancer Individual susceptibility to obesity ls strongly in uenced by heredity These mice are the same age Both are homozygous iar a defective variant of leptin The mouse on the right received daily But at in But most obese humans do not have a deficiency in leplin More than 2 other genes have been associated with obesity Leptin and adiponectin convey signals of nutritional excess blood Ghrelin and PYY convey shortterm signals of hunger or satiety Ycu39re full S You re hungry Ea top eating Emu W J Marx CeHularwarrierS at me name 01 the bulgequot Science 299 846 2003 ED osen M piege man Adipocytes as regulalors or energy balance and glucose homeostasisquot Nature 444 847 2006 17 Cholesterol amp lipoproteins Cholesterol makes phospholipid membranes more plastic 3 I E with a cholesterol 395 l E l Ho 43 l E I d cholesterol 5 Wlthout I cholesterol Temperature gt Cholesterol associates with sphingolipids and certain proteins to form rafts in the plasma membrane lipid raft sphingolipids amp other phospholipids other phospholipids ill protein with glycosyl phosphatidylinositol K GPI anchor at carboxyl end Iv cholesterol n l Lipids probably are more ordered in rafts than elsewhere in the membrane because sphingolipids usually have long saturated fatty acid side chains Cholesterol serves as a precursor for steroid hormones amp bile acids 0 ChOIeStem39 Qltg Egesterone HO OH testosterone Sjtradiol O HO CHZOH OH cortisone O HO Steroids are members of a large group of natural products with structures based on isoprene squalene H0 30 carbons Bcarotene 40 carbons Experiments with radioactive tracers showed that rats synthesize cholesterol and squalene from acetate CH3C0239 gt squalene gt acetate H O cholesterol The labeling patterns in squalene and cholesterol were similar supporting the hypothesis that squalene is an intermediate in cholesterol biosynthesis The 5carbon isoprenoid building block is synthesized from acetate by way of a 6carbon intermediate mevalonate CH3 CH3 HO CH3 C C 00239 gt gt gt H C at w it t2 i 2 it 002 002 239 CHzo 6 CH20 three molecules mevalonate isopentenyl of acetate pyrophosphate Mevalonate is synthesized from three molecules of acetleoA via BhydroxyBmethylglutarleoA HMGCoA HMGCOA CH COASH CH COSCoA CO I u i C H 2 2 C H 2 CH3 CH3 CH3 CoASH l I CO 39 quot0A COSCoA COSCoA ECOESCOA W 2 acetleoA acetoacetleoA 2 NADPH 2H COASH reductase CH2 CH2 l key control point 00239 for Cholesterol biosynthesis mevalonate Formation of isopentenyl pyrophosphate from mevalonate consumes three molecules of ATP HOCCH3 HOCCH3 HOCCH3 CH2 CH2 gt CH2 in I CH2 CH2 002 H20H coz CH20 coz CH20 mevalonate ATP CH3 OiCCH3 Pi I3 lt3H2 ltsz HZC ltin quotuquot cozy CH20 2 CH20 vhf isopentenyl pyrophosphate lsopentenyl pyrophosphate tautomerizes to dimethylallyl pyrophosphate which can form a relatively stable carbonium ion f 3 ii C gt C lt HZC CH2 H3C CH 39 a CH20 CH2390 isopentenyl pyrophosphate dimethylallyl pyrophosphate Ho CH3 CH3 C HZC CH2 H HZCquot39CH2 allyl carbonium I ion CH2 CH2 lsopentenyl pyrophosphate and dimethylallyl pyrophosphate combine to form geranyl pyrophosphate dimethylallyl CH3 pyrophosphate CH20 isopentenyl pyrophosphate CH3 Ho 39 c H3C CH H c 2 CH2 C CH 393H20 geranylpyrophosphate 10 carbons H3C This creates a new allylpyrophosphate derivative that can combine with a second molecule of isopentenyl pyrophosphate to form farnesyl pyrophosphate 15 carbons Two molecules of farnesylpyrophosphate condense headtohead to form squalene NADPH 2 O39 Formation of squalene epoxide requires 02 and NADPH H20 squalene H NADP squalene 23epoxide squalene monooxygenase Cyclization of squalene epoxide zips up the sterol rings Protonation opens the epoxide and generates a carbonium ion that reacts with the nearby CC double bond creating a new carbonium ion I HQ gt HO there s more The cyclase reaction continues 0 H0 Hydrogen atoms 0 jump between adjacent carbons and then o o g HO HO 1 HO methyl groups 0 jump and then a proton departs and we have lanosterol I won t expect you to remember these details Cholesterol biosynthesis is controlled primarily by HMGCoA reductase BHydroxyBmethyl glutarleoA Phosphorylation lt insulin inactivates HMG HMGCoA reductase CoA reductase lt quotquotquotquot quot glucagon dephosphorylation m ev al 0 n at e activates it 4 l enzyme enzyme l synthesis proteolysis l Synthesis of the lowdensity 3 cholesterol quoti i r teiquot LDL recent Whict mediates uptake of lipoproteins Cholesterol or a cholesterol derivative containing cn iester0is also is inhibits synthesis and stimulates regylated Choestero ore proteolysis of HMGCoA reductase deriVatiVe inhibits synthesis 0f the receptor Cholesterol prevents activation of transcription of the HMGCoA reductase gene ER membrane 055 SREBP Cholesterol binds reversibly to a protein that holds the Sterol Regulatory Element Binding Protein SREBP in the ER membrane In the absence of cholesterol the proteins separate and SREBP is cleaved by proteases L gt to nucleus A soluble fragment of SREBP diffuses to the nucleus where it activates transcription Lipoproteins carry cholesterol between the liver and other tissues plasma membrane monolayer of phospholipids amp some free cholesterol core of triacylglycerols amp cholesterol esters apolipoprotein LDL LDL receptor This is a model The structures mood of lipoproteins are not known cytosm CeHstakeupIOMFdens y lipoproteins by receptor mediated endocytosis a V K may m M Golgi WK 7 K Kr receptor 7 J resyntheSIs C endosome u uquot ER n cholesterol 3913 K W a 4 lysosome w a 7 nucleus gt amino lipids ac39ds Partial structures of ApolipoproteinE and the LDL receptor are known Receptorbinding domain of ApoE ApoE the only protein in most LDL s has 3 common alleles ApoE3 is the most common form ApoE4 has been linked to Alzheimer s 39 disease amp elevated risk of heart disease quotpoproteimbinding ApoE2 binds poorly to LDL receptors amp is portion 0f the LDL associated with hyperlipidemia receptor Lowdensity lipoproteins LDL and VLDL carry cholesterol from the liver to other tissues high density lipoproteins carry it in the other direction VLDL LDL HDL Density glmL 095 1006 1006 1063 1063 1210 Composition wt protein 10 23 55 phospholipids 18 20 24 free cholesterol 7 8 2 cholesterol esters 12 37 15 triacylglycerols 50 10 4 High levels of cholesterol in the blood can result in atherosclerosis deposition of cholesterol and other materials in the inner walls of blood vessels Coronary artery disease is the leading cause of death in industrialized countries Major risk factors ohigh blood cholesterol especially LDL cholesterol gt 100 mgdL osmoking diabetes mellitus oobesity ophysical inactivity There usually are no symptoms until blood flow to the heart is seriously compromised atherosclere f ltii plaque Blood clot Individuals with defective LDL receptors have exceptionally high plasma cholesterol familial hypercholesterolemia Because cholesterol does not enter their cells HMGCoA reductase is not regulated properly and cholesterol biosynthesis remains switched on If untreated people with this condition tend to die of atherosclerosis at a young age Inhibitors of HMGCoA reductase statins are used clinically to decrease cholesterol biosynthesis R1 R2 H H Compactin CH3 CH3 Simvastatin Zocov H OH Prevastatin Prevacol H CH3 Lovastatin Mevacor mevalonate In addition to decreasing LDL cholesterol statins decrease the level of Creactive protein in the blood Creactive protein is a marker of acute inflamation Its role in atherosclerosis is unclear Ridker et al New Engl J Med 325 20 2005 Nissen et al New Engl J Med 352 29 2005 amino acids 8 l Nitrogen cycles between oxidized at reduced forms in the biosphere synthesis degradation reduction microorganisms ammals amp plants is some plants is animals microorganisms anaerobic bacteria denitri cation mm xation nitrate gt gt ammonium anaerobic Rhizobium 8t bacteria some other bacteria nitrification nitrification Nitrobacler at Nltmsorncnas amp other soil bacteria nitrite other sail bacteria more reduced in the industrial Haber process N is reduced to NH by H at high temperature and pressure With an iron oxide catalyst hi2 3H1 from cm The reaction Is exothermic by 924 lemol at standard temperature at pressure but has a very high activatio ergy 2 NH3 The roots of leguminous plants have nodules that contain Nzfixing bacteria Bacteroids rodlike bacteria containing nitrogenase live inside the nodule cells nodule cell nucleus baclemids Nilragenase is very sensitive to 02 It is protected in the nodules eghemngiobin a heme rotein with a strong 1 by the plant acterial respiratory chain keeping the 02 concentration ow Nitrogenase from Azabacter vinelandii has ironsulfur and ironmolybdenum centers FeMo protein L MoFeS cluster Mo7FeSS 8Fe75 cluster 4FeAS cluster Mg ADP 2 the two ATPbinding sites are WC p6 structurally homoiogous m G pmleins I CHIC Cys residue The FeMo cofactor T me mm contains homocitrate reaction is not known Homocitrate Svhydrony carboxyadipic acid I won39t expect you to remember this structure Nitrogenase uses 8 4 c A 4 co2 electrons and 16 ATP 4 pyruva e 4 acetyICoA to reduce N1 2 H39 o 2 NH 4 H2 The Fe protein translels one electron at a time to the FeMo protein quotiiquot ced FeMo proteln redu The ATP stoichiamatry is uncertain On 5 ATP are needed under some conditions The rst step in catabolism of most amino acids is transamination go co H3NcH 20 amino acid k It uketo gt lt acid co I 0239 420 HJN Ir H iquot 6quot in 6quot co col glutamate aketoglutarate into a small number of amino acids particularly Glu a Aspt Some amino transferasas transaminases are speci c for uketoglutatale and Glu others use exaloacatata and Asp Transaminases use pyridoxal phosphate as a prosthetic group CHZO H20 CH0 L sNH oCH NH l L NH V 2 l i v y HO CH H10 H0 CH3 pyridoxal phosphate Pyridoxal phosphate forms a Schiffbase aldimine bond to a lysine residue 0 t e en This reaction is readily reversible CH0 CHZOH HNCH um HDCH NH Cl39 3 H CH H0 CH3 Pyridoxal phosphate transfers the amino group by shuttling between aldehyde and amine forms H amino ozc b R1 39OzC O R 9 acid 1 gm 5 acid1 CHD CHZNH Ho CH20 HO C z 39 I pyridoxamln phosphate CH N on 3 phosphate on enzyme H on enzyme H gt amino 39ozc b R1 39oc o R2 uketo acid 2 m 5 sold 2 enzyme This Is a classic pingpongquot enzyme mechanism The positive charge of the pyridoxine ring facilitates an interconversions of Schiff base intermediates H al ate pyridox Cquot L phosph 5m 7325 5 R 39ozc C R I a acd 3 H20 u g 02 III R N N r V CH2NH1 LL Hz 4 2quot HO quotz 39 HO EmuB Ho CH3 N pyridoxamine Cquot N Schm CH N M phosphate base 3 H nal residues that ng and release The active site has addi could fac tate proton bmdl Schi base armed ham PLP amp ZmethylAsp aspartate aminotrznsferase lalspdb Related enzymes use pyridoxal phosphate to catalyze amino acid racemizations and decarboxylations H The amino groups of glutamic acid and glutamine can be released as ammonia in liver mitochondria 02 transaminases 3920 I co a keto amino IN I H acids acids R In 30239 c HZNCH 392 L39H Ik t h I 2 2 er en CH2 I39mZ Glu glutarate I glutamate I 2039 CD dehydrogenase 80239 39 Z HINFH NADH or Pquot NADPH H NAD or quot20 CH2 NADP I39IDNH Z Butammoniaistoxic G I a cuIarIy to neural tissue m m n rlon musc e other tissue Terrestrial organisms must prevent it from accumulating Amino acid decarboxylases generate AmImonl39a Ifhmmrsorlatted mt mamI btmlqglcal amines that serve as neurotransmitters mo ecu es mug 9 u ama e aquot 9 H mm 02 C CD co CD CD I r 1 l 1 I 2 39ATP ADPP I 2 KN IF MIN HJNELH ol HNH W4 1 H3NCIH cH2 1 SshydmxyTrp 2 CH2 CH2 CH2 Glu I dihydroxyPhe I I I 3quot IDOPA quot0 H on v V CH1 2 H o ciiz Gln co H H 0 NADH NAD IIIO 2 I A 2 or or 1 G39 0 2 902 ex coZ 4 CO2 NADFH NADP 3 a keto 39l39Hn 39I HJ NH glutaraie 1 ltH2 I 2 3 C02 CH CH CH Li H1 0f a keie I 1 Ho H Glutamate dahydmgenase 1 CH1 Elmira 801 DH and glutamine synthetase z I yaaminobutyrate 0 are found in all organisms CH2 GABA dopamine serotonin Reaction 3 occurs in plants a bacteria but not ani alsl CO2 Also Histidine a histamine 002 Glutamine synthetase catalyzes formation of glutamine from glutamate and NH co foz H I 2 HJNcH MIL EH2 ATP ADP f CH H2 LA I 2 co 096390 The reaction proceeds through an enzyme bound yglutamylphosphate intermediate Glutamine serves as a donor of amine groups for synthesis of many other molecules i In most terrestrial animals Gln also carries ammo 39 nIa In kidneys where it is hydrolyzed for excretion as urea urea 4 NH 2 CTP carbamoylvphosphate glucosamineEP 39 alanine gt glycine gt histidine J 39 tryptophan AMP Glutamine synthetase is inhibited allosterically by many of the endproducts GIquot glutamine coi synthetase ATP ADP 39HzNCd l NH P OH I 2 cHZ 0 Q 0 co A A 4 4 The inhibitory e ect of all the pruducls acting together is greater than the s m at their individual elfeclsl Bacterial glutamine synthetase has 12 identical subunits 09 quot1 gt histidine carbamoyl Gln phosphate V glucosamine 0 sP gt glycine views of the Salmonella typhimurr39um parallel and perpendicular to the 6fold symmetry axis Zgls pun E cali glutamine synthetase also is controlled by covalent modi cation ATP PPl mmm adenylylatlon g39mamine Mi 0 synthetase OAMP deadenylylation inactive ADP P a keto g utarate adenyl group 0 II o f ocnzwaem 0 muscle protein Alanine carries amino groups from muscle to the liver for excretion glucose amino acids aketo glutar 0 c o EH3 pyruvale ale 02 39HJN l H Ala CH aketo glutarale
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