BIOCHEMISTRY BIOC 440
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This 31 page Class Notes was uploaded by Hertha Kihn on Wednesday September 9, 2015. The Class Notes belongs to BIOC 440 at University of Washington taught by Staff in Fall. Since its upload, it has received 16 views. For similar materials see /class/192392/bioc-440-university-of-washington in Biochemistry at University of Washington.
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Date Created: 09/09/15
The Molecular Logic of Lifequot I What is Biochemistry II Unifying Themes in Biochemistry Assigned reading Chapter 1 p 1 2 Section 13 p 19 27 Review Section 12 Homework Problem Set 1 BlOC 440 Lecture 1 1 Chemical Concepts You Should KNOW andor REVIEW General Chemistry Concepts covalent versus noncovalent bonding hydrogen bonding ionization of HZO pH strong acidsbases disociation constants simple chemical kinetics N Organic Chemistry Concepts functional groups what are they what can they do conformation vs configuration chirality stereoisomers Simple Math how to take logarithms in your head See Logarithm Primerquot on BIOC440 Web age Units see Review of Unitsquot on BIOC440 Web page Iquot 2 What IS Biochemistry Biology Chemistry Biochemistry Paradox Molecules are lifeless Yet all of life as we know it is due to the properties of the molecules of lifeg Unifying themes of Biochemistry 1 CELLS are LARGE compared to molecules I Diameter of a liver cell 50 um I Diameter of actin large protein 40 1 Cell diameter protein diameter Like a 2m luman living in a room with I Cellular concentration of any specific molecule is LOW 1o12 103 M I But of a specific molecule is still HIGH EXAMPLE glucose 1 X 10393 M Moles glucose 1 X 10393 molL glucose molecules So biochemistry follows Laws of Thermodynamics Allows biology to quotpredictquot behavior of molecules 4 BIOC440 Lecture 1 IH Unifying themes of Biochemistry Unifying themes of Biochemistry 2 LIFE opposes ENTROPY 5quot 2 LIFE opposes ENTROPY b Entropy amp transformation of matter oxidation of glucose 2nd Law of Thermodynamics a Entropy amp energy heat exchange C leo 6 02 gt6 602 6 H20 25 C quot7 lt 725 C of atoms ll 734 of molecules g K A y 100 oc j i i C states Free energy AG39 25 C quot 25 C 37quotquot 45 C Living systems take the free energy and release the products and entropy to the surroundings 5 6 Unifying themes of Biochemistry Unifying themes of Biochemistry 2 LIFE opposes ENTROPY 2 LIFE opposes ENTROPY c Entropy amp information c Entropy amp information 139 i Jim I I 2 f Jim I 3 There is a tide in the 6 r g P n Q 39IS I f s a g affairs of men a 63 x 99010quot S a 83 o 904 quot whichtaken at the flpod quot 4 9 DJ 0 jagefqh mus 9 leads on to fortune ef Q39MomS aglj h e n weh Q39Mom 339u h Living systems are highly ordered amp nonrandom ORDER requires WORK and ENERGY input BIOC440 Lecture 1 Unifying themes of Biochemistry Unifying themes of Biochemistry 3 LIFE requires ENERGy 10 Pay for 5 4 Living systems LINK biochemical reactions unfavorabh ego ve enfropy MANY biochemical reactions are not spontaneous Endergonic process living systems are NOT at equilibrium w Exequot9 quoti Pquot SS wrroundings a Methanical example AGgt0 AGlt0 living systems TRANSFORM energy into work to x maintain order m quot Fuzzy raising x s energin posltlon 9 Endergonic Exergonic LEM 10 Unifying themes of Biochemistry Unifying themes of Biochemistry 5 Biochemical reactions are CATALYZED to proceed at 6 Biochemisfr y occurs in AQUEOUS medium useful rate the properties of H20 influence all of biology Review reading Chapter 21 Activation barrier transition state t A63 Reactants A T AG fat Products B Free energy G AG Reaction coordinate A gt B Fig 1 27 11 BIOC440mee1 12 BIOC440 Lecture 1 Protein Foldingquot I Protein denaturationrenaturation II Proteins helping proteins Chaperonins III When proteins go badfolding amp disease ASSIGNED READING Chapter 4 Section 44 Section of quotcircular dichroismquot p 122 amp article on chaperones Web link on BIOC440 Schedule page HOMEWORK PROBLEM SET 3 B IOC440Lecture 7 1 How stable is the folded protein 1 Study wfolding denaturation Folded FlNative N gt UnfoldedUlDenatured Unfold the protein by adding Ribonuclease A Measure 0 property that changes bet folded and unfolded state Apnmyuglohir Percent of maximum signal 20 40 so so Temperature quot2 E a Calculate amp plot fraction that is unfolded versus additive or temp Percent unfolded l 2 3 b GdnHCl M Figure 25 Can proteins fold on their own Anfinsen Studied protein kfolding of purified RNAase Native RNAase has 4 55 bonds w UNIQUE pairings 72 53 65 95 ylkall on additionunread denaturant unfolding agent mmapmuhano BMEH reducing agent 12 H5 Unfolde me 5quot inacliveisulfide nksredmedlo c yield cysyesidues removal olurea and meraploElhanol 72 59 65 Name m N 26 caiflwlcally azllvestaie 95 DisullidecrassIinks 0 orrecilyrelnrmed Figuum 43925 3 HOW Do Proteins Fold E Coli synthesize ACTIVE folded protein in lt5 seconds N amp Ctermini are often close in 3D structure implying folding cannot be simultaneous with protein synthesis Levinthal39s paradox Too many options For a 100residue protein assume 10 possible conformationsresidue ie rotations about single bonds possible conformations to be tried to find native one If each try takes 103913 s time of a bond vibration Proteins must fold via NONrandom pathway4s Somemost proteins need HELP Protein misfolding 15 bacterial proteins do not fold correctly on their own Molecular chaperones protein folding helpers 1 gt F U 12 gtMisfolded 13 A re ate IN 99 9 Proteins helping proteins 1 Heat shock proteins HSPs abundant in cells under stress conditions high temperature low pH etc Molecular life raftsquot DnaJ amp DnaK are bacterial HSPs Dna binds lo ihe DnaJ stimulates m unfolded or panially 1 Pi hydrolysis by DnaK aided pvoleiu and DnaKADP hindsiiglnly quotmu m DnaK m die unfolded pnnein j 71 e Inquot l v e 4 Unlolded Ln protein To GmEL Panially system 5 NI Inldeg pmen i 3932 ii39 vaE Folded l 7 protein 1 l l lnative r L conlormnuon lT ADP tarps anal n l fl ATP bininn m In baaevimhe DnaK and IE nutlenlldeexthan 2 protein dissociates lactorGrpEstimulates release anDP Figure 4 29 5 Proteins helping proteins 39 l H I 2 Chaperonlns day spa for proteins multisubunit complex forms vessel w cavity big enough for unfolded chain Unfolded l l Un39 drd 91min The released pmteinbindsl Gran n E quotS ulsinuirsfxl ly 3232 Lit 1 v r 39 l E Q 6 2f liquot n minim E Pquot quot Ihh5l ll 3 7W Dzildedwnen f7m 2 4 232 nflheGroEL l releasedare I 7 quothi 7 l 7W l 7 ma a 1 7AYPandeaE5 blndlo mELwlih a lled pnekei Figure 4 30 7 Structure of a Chaperonin GroELGroES Surface representation Slice through middle Figure 4 30 b cavify When profeins go BAD Bad Things happen Protein Misfolding Diseases Alzheimer39s Huntington39s Parkinson39s mad cowquot w Native Molten globule Denatured it w afte r Selfassociation 5 ail Amyloid bril or structure Further assembly of proto laments Figure 4 31 a BIOC440Lecture 7 9 Biochemistry 440 Fall 2008 Lecture 24 g Oxidative Phosphorylation I Energy balances Oxidative Phosphorylation 1 Biological Redox Reactions I Reduction potential biological redox reactions I Electron carriers I The Electron Transport Chain Reading assignment I Elucidation of the Electron Transport Chain Chapter 134 p 512 521 I ATP synthase Biochemistry 440 Fall 2008 Lecture 24 Balanced equations for glucose and a fatty acid ogddation pathways I Glucose 2NAD ZADP 2Pi gt 2 pyruvate ZNADH 2H 2ATP ZHZO I Pyruvate NAD COA gt acetleoA CO2 NADH H Acetyl CoA 3NAD FAD GDP 131 2H20 gt 2co2 3NADH 3H FADH2 GTP CoA I Palmitoyl COA 7C0A 7FAD 7NAD 7H20 gt ADP 02 8 acetyl COA 7FADH2 7NADH 7H ATP H20 Biochemistry 440 Fall 2008 Lecture 24 Balance sheet Glucose oxidation i Balance sheet Palmitate oxidation Biochemistry 440 Fall 2008 Lecture 24 Electromotive force AE Reduction potential E I Electrons are spontaneously donated from species with low electron af nity donors to species with higher electron af nity acceptors gt favorable process One way to consider the potential energy of electrons I Reduced donor Oxidized acceptor I A measure of the tendency of a chemical species to be reduced or D quoterg oxidized I Oxidized donor Reduced acceptor I Low electron af nity gt I Driving force behind the electron ow Electromotive force AE gt I AB is the difference in reduction potential E between electron acceptors and donors gt 39 AB Eacoeptm39 39 Ednnm39 I AB is related to AG free energy change of a redox reaction gt HOW to quantify AG nF AE n number of electrons transferred F Fara day s constant 965 k Volt391 Inol39l I High electron af nity gt Biochemistry 440 Fall 2008 Lecture 24 Measurement Of reduction potentials Reduction potential compared to a reference cell Redox reaction XCDlt B ed ltgt X ed BDgtlt XCDlt electron acceptor B ed electron donor Written as two half reactions reductions nHt ne39 ltgt BDX nHt nee ltgt B ed salt bridge XCDlt and X ad as well as B ed and Bax are redox couples or redox pairs voltmeter Standard conditions 1 M everything Compared to a reference 2H 2e lt gt H2 arbitrarily set at zero If E0 V negative BDXnHtnee ltgt BM anthltne39 lt gt Xred If E0 V positive salt bridge Biochemistry 440 Fall 2008 Lecture 24 Hi The biochemical standard state pH 70 5 Some Standard Reduction Potentials Acceptors 7 Donors E 0 V E 1 Meverything except 1 x 10 7 M H NOTE The reference is the same 39 12 02 H20 032 l FAD FADH2 003 l NAD r NADH 032 a ketoglutarate isocitrate 038 For a given redox couple eg NAD rNADH Negative standard reduction potential E gt Negative standard reduction low electron af nity potential for the redox couple Xox Xred Positive standard reduction potential E high electron af nity salt bridge Biochemistry 440 Fall 2008 Lecture 24 The difference in standard reduction otential AE O is used to analyze AG 0 The actual reduction potential E depends on concentrations rof e39 donors and acceptors Whole reaction Whole redox reaction H H cti 39 NADquot isocitrate ltgt NADH H r ocketoglutarate COz x fenaH ltJgt X ed acceptor X B ltgt X B m ad ad BX BDX nHt nee ltgt B ed donor Two half reactions AE D V ocketoglutarate COz ZH r Ze ltgt isocitrate 038 NAD 2e 2H lt gt NADH H 032 gt BMW and Edam from 2 303RT X NernstEguation E E 0 39 log ox Which is the electron acceptor accePtor quotF del What are AE 0 and A6 for this reaction 2303RT B l 0 ox E 0 E 0acceptor 39 E Udonor donolr E Tlog Bred 0 7 0 G 7 HF AE AE Eacceptor Edonor nis the number of electrons F is FaradaYs constant 965 k Volt 1 rnol391 R is the gas constant 8315 K71 rnol391 Tis absolute temperature AGnFAE Biochemistry 440 Fall 2008 Lecture 24 NAD and NADP are soluble FAD and FMN are enzyme electron carriers bound electron earners isoalloxazine ring o T 039 T 0 CH3 N He39 CH N He39 H N o o o 3 H II 90quot 1 c 29 c C ms N N 0 CH3 N N 0 cu N N 0 H2 quotHz or NH m I I 3 Cl R R H 0 012 0 N 2 quotf T FMN HCIOH FADH39FMNH39 FADH2FMNH2 I H H R A side R B side co semiquinone fullyreduced opo H H NADH HCIOH I 0H 0 reduced cl T NH FAD I OP O N Adenine 39O I O I l J o N quotquotquot quot l quotquotquot quot NH2 o CH2 0F0 N H H I N l J NAD H H g o N N oxidized OH OH 2 H H In NADP this hydroxyl group H H t 39f39 d th h h t 0H 0H Is es en Ie w P osp a e Flavin adenine dinucleotideFADand flavin mononucleotide FMN F1gure 13 24a 15 Lecture 17 F1gure 13 27 16 Biochemistry 440 Fall 2008 Ubiquinone Q Electron carriers EHS 30 CH2 CHC CH2m H Ubiquinprre Q I CHSO CH3 fully ox1dlzed 0 ll H I 039 CH30 R Semiquinone radical QH39 CH30 CH3 I OH l P I 39 9 H CH30 R Ubiquinal QIIZ fully reduced CHgO CH3 0H F1gure 192 17 Lecture 24 Cytochromes Electron earners S Cys l Cytochromes cu chxIz Cys S I CH3CH CH3 I CH3 CH2CH2COO39 CH3 CH2CH1C00 Heme C I in c type cytochromes CH 3 CH C H I OH CH3 Hz CH CH3 CH3 CH3 CH3 CH3 CHZCH ZCOO39 in a type cytochromes CHO 042042500 Figure 1 93 18 Biochemistry 440 Fall 2008 Electron carriers IronSulfur Proteins a b Cys akO Cys 3 Cys 9 Cys Cys Protein Figure 19 531 Lehm39nger Plinriple of iorheminry Hm Edilian m 2008 w liFveeman and Company Reduction potentials vary from 065 V to 045 V depending on microenvironment in the protein Fig 195 Lecture 24 10 Protein Structure IV Tertiary amp Quaternary structurequot I What stabilizes tertiary structure II Fibrous proteins III Globular proteins IV Quaternary structure ASSIGNED READING Chapter 4 Section 43 section on symmetry optional Lehninger Web tutorial use Web link listed on BIOC440 Schedule page HOMEWORK PROBLEM SET 2 BIOC440 Lecture 6 1 What stabilizes 3 structure Forces discussed in lecture 5 1 2 3 What stabilizes 3 structure Special cases 1 Disulfide bonds covalent bond bet 2 oxidized Cys residues Found primarily in extracellular proteins Can be intrachain or interchain insulin What stabilizes 3 structure Special cases 2 Metal binding stabilizes some intracellular proteins reducing environment so no u u disulfides zmc fmger mo l39lf Bioc440Lecture 6 Two classes of proteins Fibrous amp Globular 1 Fibrous proteins form STRONG amp FLEXIBLE structures Strumlre havactetistic Examples of nmmeme a Helixcrosslinked by 1quot and naik B Conformation Soft flexible filaments Silk fibroin Collagen oftandons hone malvix Fibrous class of proteins Collagens many types usually contain 35 Gly 11 Ala 21 Pro 4 Hyp amino acid sequence is repeating triad Gly XY w X usually Pro amp Y usually 4Hyp 2 structure collagen helixquot w 3 residuesturn coiled coil of 3 helices m Figure 4 42 Globular class of proteins 2 Most proteins are globular 3D structures are determined experimentally C atomic level very large proteinscomplexes requires a protein CRYSTAL requires strong xray requires fast computers Bioc440Lecture 6 Xray Diffraction or Protein crystallographyquot SOUPCB p 132 33 crystal structurequot 8 Protein NMR spectroscopy Different ways to represent structures requires large ribbon spacefillingquot 31 amounts of soluble quot protein but no crystal requires high q quot bu amp s ckquot m magnetic fields 7 i P superconducting 3 magnets j i requires fast quotsurface quot compufers representation m P 13334 solution structurequot Figure 415 10 Gen39l features of 3D structures Gen39l features of 3D structures 1Types amp arrangements of regular 2 structure 3 core and surface aa type distribution 4 Amphipathic helices amp sheets allow for 2 core and surface efficient packing of hydrophobics In general helices amp strands comprise the core turns are on the surface 11 Figure 4 170 12 Bioc440Lecture 6 Gen39l features of 3D structures Domains Quaternary Structure Multichain proteins 5 Many proteins are MODULAR and fold into Each polypeptide chain quotsubunitquot independent domains that retain structure amp function multimer or oligomequot Hemoglobin tetramer of 2 a amp 2 Bchains If chains are all the same sequence 3 qu calmodulin Figure 418 13 14 Quaternary Structure Interactions between subunits are often hydrophobic This poses a challenge Fig 4 2 b BIOC440 Lecture 6 15 Bioc440Lecture 6 Twicks to remember Twptophan is quotWquot and other amino acid code hints Professor Klevit39s helpful hints for remembering 1letter amino acid codes No hint means it39s the obvious choice 3letter 1letter code Hint Ala A Cys C Asp D quotasparQatequot Glu E quot lugquot Phe F quotEenylalanine Gly G His H lie I Lys K quotKlysinequot almost sounds like quotglycinequot Leu L Met M Asn N quotasparagi equot or quotAsN Pro P Gln Q quotQ taminequot Arg R quotB ganinequot Ser 8 Thr T Val V Trp W quotthtophanquot Tyr Y quotI rosinequot Biochemistry 440 Fall 2008 Pyruvate Dehydrogenase Citric Acid Cycle and Glyoxylate Pathway Chapter 16 p 615640 Glyceraldehyde 3 ho sphate ADP Electron Transport and 02 Oxidative Phojsphorylation Rllmsumcs lmwr mwnin39mw f Hirl nnvmhmnr Lecture 21 The bridging step Pyruvate gt acetleoA I Pyruvate COA NAD gt acetyl COA CO2 NADH An oxidative decarboxylation catalyzed by Pyruvate dehydrogenase C02 K o CoASH TPB c NAD Iipoate NADH o 5c A I c i0 pyruvate dehydrogenase I Ha complex E1 E2 E3 CH3 Pyruvate AcetylCoA Figure 1672 AG 334 kJmol Figure xsz unnmymmpmarmmm mam Lecture 21 Lecture 21 Biochemistry 440 Fall 2008 Lecture 21 The PDH complex P ruvate Dehydrogenase Complex mm m z Enzyme prosthetic group substrate E1 TPP pyruvate E2 lipoic acid CoA E3 FAD NAD 9 5 CoA SH H g 5 c A c 39 3 39 0 I 0 c o K AcetylCoA CH3 C C Pyruvate o lipoyllysine Figure 166 C02 NADH H Hydroxyethyl TPP Oxid39zed NAD 5 Lemme 21 llpoyllysme 6 0 39d39 d R d d A l d Step1 Pyru vate d ecarboxyl ati on 29 23quot in 21 Steps 2amp3 Ox1dat10n and CH1 HS Hg CH3 C S CH2 m cuz gtCH2 transfer to acetylCOA quot Figuresl414 w quotHt quot Q Lipoj CH2 fquot Kquot Coenzvme A CoASH NH C5 and 166 and CQZ c HI N z 2 1 O 0 fa N II II cg x 5 CHz CHz O P O P O co N CH3 L L quotN 39f T Im I 39I 539 ltM I Jquot CH3 N O O Ratellmltln Ste H C Hz CHI N C C K CHz O P O P u Hz M g P Ly 2 n l n 0 Thlamlne pyrophosphate TPP midquot cm 0H CH3 0 a n uf E2 CH2 Pinluxheni idd 3 2 H J Q1 u M m 74 N c n I th1azohumC2 10nlzat10n gt H g 3 carbanion formation gt hiPOY39IYYl E1 E2 I Ipoaml e V attack of pyruvate carbonylcarhon m oi ety of E2 0 o of 0 CDA SH CH3 c s CoA decarboxylatlon after e delocahzatlon CH gc 9 Acetyl CoA 3 0 Reduced J Pyruvate Tpp 39iPDY39IYSine Acy T 39 CD Iipoynysinel L act v Tpp ys CNS i OH aceta gme co NADH H NH2 c FAD N CH S ti ltl CH2 CH2 O P O P O39 F Hydmxyethyl J CH3 lgum TPP muquot CHa N o o 16646 lipoyllysine Biochemistry 440 Fall 2008 CH3 C C Pyruvate O u 0 O Hydroxyethyl TPP ll do CcASH CHS C SCoA Example Steps 4amp5 Lipoarnide regeneration and electrontransfer to NAD D Figure 166 AcetylCoA Redued lipoyllysine yl oyllysine f Lys NADH H PIX Iipoyllysine NAD E3 Pyruvate COA NAD gt acetyl COA CO2 NADH of substrate channeling An overview of the citric acid cycle Lecture 21 10 Acetyl CoA 0 Hsti iS COA OO CoASH 20 C t t EH COO CH2 Oxaloacetate nae 02 l H Fum arate H COOquot Succinate O EH2 HOJ JH coo 007 CH2 H2 Malate E i300 Isocitrate 06 HOAEH H10 coo OU a ketoglutarate 00 H2 EH co Succinyl COA CoASH i320 CoASH goo c0 CU H2 2 2 H2 0 woA Acetyl CoA i H3C7C787C0A ECU H e CItrate 2 Malate C00 5 nthase HO 00 39 deyhdrogenase 0 y l aconltase c o l C1trate H2 CH2 Oxaloacetate 00 l HO H 00 COCT EH2 Malate Isocitrate H2 00 H 37COU fumarase 39l HOjH CO OCT H COU Hg Fumarate OLketoglutaratg A Succmyl CoA H2 Isomtrate 00 synthetase H2 dehydro Succinate Succinate SuccinylCOA 0 genase coo deyhdrogenase j H 2 OCT ng CH 02 H2 xketoglutarate dehydrogenase 700A Lecture 21 Biochemistry 440 Fall 2008 Products frcrn the Citric Acid Cycle From 1 round of the cycle 3 NADH 1 FADHZ 1 GTP 2 CO2 released 1 NADH from bridging step Lecture 21 Regulation of the Bridging Step I Pyruvate dehydrogenase I Inhibited by I Activated by Regulation of the Citric Acid Cycle I Citrate synthase o Inhibited by citrate NADH succinyl COA ATP I Activated by ADP I Isocitrate dehydrogenase I Inhibited by ATP Activated by Ca2 ADP I ocketoglutarate dehydrogenase I Inhibited by succinyl COA NADH I Activated by Ca2 Lecture 21 Lecturell 14 AcetleoA i cocr Hat cg H2 HO 700C 0 H 0001 iomomme cm 2 M H07 H ECU ioo EH2 Malae Isocitrate H2 coo HAticocr Ho H cocr 06 H Hi Fumarate aketoglutarateioo H2 006 IHQ N Succinate SuccinylCOA ECO cocr IH 00 00 CO2 amp 2 EH2 CO H2 EH2 e00 700A Lecture 21 Biochemistry 440 Fall 2008 Lecture 21 The Citric Acid C cle is am hibolic i Citrate is prochiral y p IZHz MCOO Anaplerotic l pymvm l reactions 14 CH COO CH3 C00 I Labeled acetate CH2 coo39 HO CH COO carboxylase AcetyICoA PEP carbaxykinase I Phospho glpyruvate EP Oxaloacetale 39 b c car oxy ase cilrk OC CH2 ycgle rxKetoglutarate CH2 coo Labeled citrate CH COO Oxaloacetate CH C00 SuccinylCoA 1 coo Pyruvate HO C H lsocitrate Figure 1 615 7 Acetyl CoA O The Glyoxylate Pathway Hacilisimm Cfooi COAS 007 H OH I Plants fungl and some bacterla can convert acetyl NS ESJF H ijooxaloacetate HOQOU CoA to oxaloacetate animals cannot go Citrate Hz 000 ooor HomH 7 I In plants this pathway occurs in the glyoxosome gHz Malate 30 a membranebound speci c planyorganelle 09 gH e00 l CoASH COG CgtHZ In bacterla and fungi it occurs in the cytoplasm 1300mm W ooe HomH i oocr HaciCiSiCoA Lecture 21 19 Biochemistry 440 Fall 2008 Balanced equation of the glyoxylate cycle 2 acetleoA NAD 2 H20 gt Succinate 2 COA NADH H Figure 16 22 Fany acids Ace lCDA OxaIuacetate r Glyoxylule Malate yde Citrate Sucrose Glyoxylale I M soduaxe 7 AcenyLCuA Hemes Succinate Glyuxysome glucuneagenesis Oxaloacelate Cymsol Malale A Fumarate Mame Citric Suuinme quotquotd Oxaloammte cycle Citrate Mihchondrion Lecture 21 Glycolysis HOCH2 H H H Glucose O OH H OH OH o l 390 o CH2 I Glucose CIH H H 6Ph0sphate OH H OH OH H l CHZOH 0 0 3 O P O CH Fructose I 6ph0sphate H HO OH l 9 3 O P OCH H C 0 0 Fructose C 2 o 2 16 bisphosphate H OH OHH CHon CH239O P O Dihydroxyacetone Glyceraldehyde Phosphate 3Ph0sphate O H C HI3OH Glyceraldehyde I 9 3Ph0sphate CH2O fgt o l O Cquot I 13 Bisphospho HIOH O glycerate CH2O gl39L o i O KC039 H3 OH O 3 PhOSth I II glycerate CHz39O ELO i O O C3 9 2Phospho HI3O FO glycerate CHZOHO KC039 I 9 PhOSphoenol IJI O chr nyuvate CH2 0 CD I Pyruvate CO CH3 Enzymes III Mechanismquot I How to study enzyme mechanism II Specific examples chymotrypsin enolase IIIMechanismbased inhibitors ASSIGNED READING Chap 6 Section 64 Box 63 optional amp p 216220 Web Resources Mechanism Animationsquot in Chapter 6 of Lehninger Web site see Bioc440 Schedule page for link HOMEWORK PROBLEM SET 5 BIOC 440 Lecmne 14 1 Enzyme Mechanism What amp How Identify 1 ALL substrates 2 Cofactors coenzymes 3 All products 4 Regulators modifications inhibitors Describe 1 Temporal events kinetics 2 Reaction intermediates 3 Active site of enzyme amp functional groups involved 4 Catalytic strategies BIOC 440 Lecmm 14 2 Specific Examples Chymotrypsin a protease Catalyzes hydrolysis of peptide bonds Ri H RH H O K Ri1 I 2 l 39 39E c8Ncc SL OW lIl C COO H3N cg N I has rate enhancement gt 109 O BIOC 440 beams 14 Figure 0 l 8 Chymotrypsin Mechanism w Figure 6 19 4 4444 4m imam StepbyStep Mechanism Chymotrypsin free enlyme Interauion of Ser and His57 generates a strongly nutleopllili alkuxide ion on Ser the StepbyStep Mechanism Instability of the negative charge on the substrate carbonyl oxygen leads to peptide linkage breaking the peptide H bondThe amino leaving quot57 grou tonated by rm His iacilitating its dtsp acement D gw e serm Shortlived AA H NH ch H NH AA intermediate 0 l acylation His N Giy39 H Productt N Writ w 4 a at 0 Ser 95 lst Covalent I C CH N H AA Intermedlate KG oxyanion H k N N rsly g1 Se mo 95 Acylenxyme intermediate 2nd Covalent HEW 1539 BIOC 440 Lecmne m Infermediafe z M mw HM atetrahedralacylvenzyme wquot t W u I g quot This isarcompanied by iormalion ashort His lived negative 4 gu charge onthe quotrm 5w tarhnnyloxy Ar Hydmphnbir Vr hfn srfgtrateglnds the slit am g39bs mquot Ho 59quot x r ano eresl an exert a V lhwmidghmdm h km Stiblllled by AAn EH NH H NH AAquot mquot H nestlesinahydmphobitpockel MP9 bquotquotquot39 o n a A k onIheenzymemositlonlnglhe Insmthe r OxvMunhnle ly39 sum pepllde bond loramtk wankquot M39s 39 N N ES complex Gly Ser speCIfICIty u pocket F5 r 23L BIOC 440 Lecmne 14 9U 2 6 S H H c Acylenzyme intermediate HisS7 3rd Covalent An Incoming water molecule quotvquot is deprotonated by general Infermedla l39e 1 base catalysis generating a u u s is r 39 39 quot er strongly nucleophlllc oxyanlon H o H H quot hydroxide ion Attack of ShortIlved 57 2 hydroxide on the ester intermediate 0 Hquot H i linkage of the acyIenzyme deac lation 39H N N generates a second y N my sen tetrahedral intermediatewith s NU lg oxygen in the oxyanion hole again C Ser quot5 taking on a negative charge H 0 i C H N H AA 0 Collapse ofthe N N tetrahedral Gly Serquot5 intermediate forms the second product a carbohydrate anion and displaces Ser195 BIOC 440 Lecmne 14 E 9 6quot 2n StepbyStep Mechanism EP Enzymeproduct Product 2 2 COmPleX H0 CH NH AA H 57 o I IS HN F iguve 6 21 Diffusion of the 9Na second product H o Ser 55 from the active Ho c CH NH AA Chymmrypsm site regenerates quot free enzyme 0 free enzyme 0 57 N O N g ub urn mm Hydrophobic pocket mm site In H IN N Oxyamon her my 5quot BIOC 440 Lemme 14 Specific Examples Enolase Catalysis by metal ion amp acidbase 2phosphoglycerate gtphosphoenolpyruvate M9 of re L 0 H 0 H 2 quotH ch l l cil lc H M9ii xvi Enolase o H OH 0 J H0 0 HO Io H Enclose mus muzn mans 5mm I Mgz lowers pK 1 of C H by 9 LLK inductive effect Mutations Lys345Ala and Glu2116n are INACTIVE 5 le Hymnquot Q Assuming active site is pH 7 what can you say about the PK 0f K345 and E211 BIOCMOLecmm14 9 Mechanismbased Enzyme Inhibitors EXAMPLE diisopropylfluorophosphate DIFP a potent general inhibitor for Serine proteasesquot chymotrypsin trypsin etc lt 3 CH3 Enz CHz OH F P O CH Serws cl CH often but not always look I 3 like a substrate c H3C H CH3 bind in active site of enzyme DIFP form a COVALENT complex F l w enzyme i39l CH3 Enz CHZ O IIJ O CH 393 CH C H3CHCH3 BIOC MOLecmiie 14Figuf 8 13 10 Many drugs are Mechanism based inhibitors penicillin Reaction in bacteria c in cillln Sidachain Yhiazclidinering benzylpanicillinl i r g N 4 5 ii i quot l l quot lo a c n e E 5 CH Q c ml J H I I Favours i C N CH Penitlllan lt 7N nAla c 9i 3 7quot u v 39 739 WCLN Sanlam C00 quotquot9 WC mum ham N H Amcxicillin h General slrunure of penicillins v i R c N T s I L I L7 n H CNCH EH ii aiwi gi iii This I o Iii quotMum Sei oH OOH Penicillin gt01 Mi 5quot fquot f H H quotFEEquot RhCaNgtAs Cll quot i 39 c N lll m pn 52a H H quota STUCK Ifquot quot O can I W quotH quotquot quot m39 Siablydeiivanzed gt1 I NAAzlm inactive transpepkidase Classlinked vuplldogl inn l6 ll V a M Tia 6 Z v BIOC MOLecmiie 14 Fquot v quot 11 Overcoming Drug resistance Drug resistance occurs when bacteria figure outquot how to inactive a drug Drug design or discovery must stay one step ahead H E H H1oc Ccmu CNgt cs CH3 O CvNCQ H H I I N c coaii Clavulanic acid R Ser b39H coon Penicillin H 10 crion MIC C w sei mlgl ltC c M j o H H o i N C II As CH coo n c Nc c 1 H C c N CH SEr 0o H Equot J 5 OHcE o CHOH coon 39 c u gt H20 GI quot 94 N H i l t H H Nulx H V s CH3 o CHZOH C u i i V WV H c MCH 0 EH H o g H cacH COOH lnac ve penicillin mm m Lemme 14 lnadive pIaciamase
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