BIOCHEMISTRY BIOC 440
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Glucose Citric Acid Cycle NADH and FADHZ Electron Transport and Oxidative Phosphorylation NAD and FAD Binding amp Allostery The globinsquot I Gen39l description of ligand binding II Hyperbolic binding 02 binding by Mb IIIigmoidal binding 02 binding amp release by Hb ASSIGNED READING Chap 5 p 153161 Web Resources Lehninger textbook site Chapter 5 Living graphsquot HOMEWORK PROBLEM SET 4 BIOC 440Lecture 9 1 Proteins function via conformational changes gt 1 folded structure depends on conditions such as presence of a ligand different activities or on and offquot are associated w different conformations EXAMPLE Caz binding to Calmodulin CaM Caz lt 39 Ca24 39 CaM Yellow hyd rophobics 2 General Expression for ligand binding PLlt P39L K KD D Define 6 fraction of occupied binding sites occupied 9 total At what L will m 0 05 As L gt 6 gt 0 Kd 5 10 Figure 54 L arbitrary units Expression for ligand binding What does it tell us n lim vh rum3 w l n l Protein Ligand Kd M 1 X 10715 1 X 1039 0 Avidin egg white Biotin Insulin receptor human Insulin AntiHIV immunoglobulin human gpai HIV 1 surface protein 4 X 10quot Ni 1 X 10397 3 X 10396 2 X 10395 Nickelbinding protein E all Calmodulin rat Ea2 Smaller KD means KD values Q For CaM when Ca2 1x10398M what is 6 when Ca2 1x10395M what is 6 use weaker binding constant from Table 51 abovezl Oz binds to Fe in heme amp to HisE7 in protein Mb 1 polypeptide chain w 8 ahelices AH heme group His F8 t Figure 55 k O2 binding by Mb Mb 02 lt gt Mbo2 Kb 902 Because ligand is a gas express concentration as partial pressure p02 9 Define p50 as value of p02 when G 05 Finally 602 Mb HIGH affinity for 02 p50 026 kPa 0003 atm p02lungs 133 kPa p02tissues 4 kPa 9020quotquot95 602tissue 10 0 05 Mb is 02 storage protein Very high amounts found in A diving mammals VPso 5 10 Figure 5 4 p02 kPa 7 O2 binding by Hb Hb39s function delivery of 02 FROM lungs TO tissue p02lungs 133 kPa p02tissue 4 kPa Q If we design a new Mb that dissociates 02 in tissues could it function as a hemoglobin Suppose p50 Then 602tissue and 602lungs 00 OO 00 OO Oz binding by Hb a 39l39e39rramer op39l39imized BOTH for binding in lungs amp releasing in 39l39issues low p02 as 02 begins 39l39o bind Observed binding curve r302 in p02 in Sigmoidal no39r hyperbolic 10 tlssues lungs lHigh affinity 602lungs o8 state Transition from Iow to high 05 affinity state 902015942 0 04 02 Low affinity o 4 5393 1392 16 p02 kPa 7 z A Binding gt 1 ligand For N ligands expression for individual binding even39l39s PNL gt PLL gt PL2L gt PLN 1 K2 KN IF binding could occur with INFINITE coopera39l39ivi39l39y P NL gt PLN Ko KD O Limi39l39ing cases 1 Noncoopera39l39ive ie n1 6 2Asn gt gt 02 binding 39l39o Hb is HIGHLY coopera39l39ive n 3 02 release by Hb recall Thai 6 occupied139o39l39al 02 released pozin p02 in 1 n tissues lungs High affinity 039s state Transition from lowto high 0396 affinity state 0 04 L ff Q What would 02 gt t 02 wa 39quot39 y release for our designed Mb w 0 Al 239 1392 16 p50 4 kPa P02kPa Figure 3 BIOC 440Lecture 9 11 Enzymes II Kineticsquot I Enzyme kinetics why amp how II Steadystate kinetics MichaelisMenten eqn III Inhibition of enzyme activity ASSIGNED READING Chap 6 Section 63 Box 6 2 optional 39 Web Resources living graphsquot in Chapter 6 of Lehninger Web site see Bioc440 Schedule page for link self test see Bioc440 Schedule page for link Why How 54 P 39 Measure Reaction RATE via ENZYME ASSAY 39 Study Enzyme amp substrates in purified form 39 Measure how rate changes with change in S pH etc 39 Need to be able to monitor S or P as a function of me 39 HOMEWORK PROBLEM SET 5 E100 440 Lecture 13 Enzyme Kinetics E General form of enzyme catalyzed rxn single substrate rxn k k2 E S 739 E S lt7 E P I k k72 constants dSdt 1 In Viva 5 gtgt E ie E is quotlimitingquot So S is effectively constant esp early in reaction k k2 ES 39 ES 739 EP k k2 2 Initial velocity of product formation v039 v0 dPdt 3 ES is in steady statequot E S amp ES are in equilibrium k2 is SLOW compared to k1 and k1 Steady state means ES does not change w time Ie 4 Write ES in terms of measurables amp constants Total Enzyme ET E ES AT ALL TIMES from 3 ES k1Ek1k2 define KM k1k2k1 5 MichaelisMenten eqn for initial velocity dPdt v0 k2E MAXIMUM rate will occur if ET 55 so vmax k2E39max k2E1 Wthh is Finally v0 Insights from MichaelisMenten Equation 1 Km the Michaelis constantquot units M v0 VW l V0 Vmaxlsl vovmax Km 5 Km l E E E El 1 gt0 5 Vmax I I K m 51 mm Living graph for Eqw 69 Figure 6 12 6 2 km turnover numberquot rxnsactive siteunit time 3 Catalytic efficiency freq wwhich E amp 5 have productive encounters cat eff kmKM Upper limit is rate of diffusion If kmKM 2 108109 M1s1 diffusioncontrolledquot enzyme Enzyme Substrate Km M kcat5391 km Km M391 5391 Catalase H202 2 x10392 1 x 107 Fumerase fumerate 5 x10396 8 x102 maleate 2 x10395 9 x102 Inhibition of Enzyme Activity Inhibitors resemble substrates They bind to active site but no product is produced 00quot 00quot 00quot cHZ EH cle gt qHz gH COOquot 39 COOquot succinate fumerate maleate There are many natural enzyme inhibitors Inhibitors may be reversible or irreversible K El Competitive inhibitors 55 ES gtEP C3 e09 vmax Initial velocity V01M Km Substrate concentration 5 mM 9 Figure 6 w Living graph E ifi Velocity in the presence of inhibitor Definea 1 IKI V0 vmaxG39KM Apparent KM in presence of inhibitor KMQP Limiting cases I 0 a as I increases a BIOC 440 Lecture 13 10 i A Brief Review of Carbohydrates If this is foreign to you please read pp 235244 Monosaccharides l Aldehydes or ketones with at least two hydroxyl groups Glyceraldehyde is the smallest aldose O H O H C Cl l H C OH HO C H CHZOH CHZOH Lecture 17 smallest ketose i Dihydroxyacetone is the CHZOH CHZOH Lecture 17 Glucose and Fructose D Glucose D Fructose Aldose Ketose 139 H o H C OH 1 l 1 H c OH C 0 2 2 HO 3C H HO sC H H 4C OH H 4C 0H H 5C OH H rC OH Lecture 17 g Glucose Can Cyclize a I lC 0 l 6cmou 6 H 23 0H cuzou H0 quotC n 13 II E 40 C ll C OH Q v 0 quot 3 2 6cugou H OH uGlucoso aDGlucopyranosc linear form Haworth projection Lecture 139 D 4c 0H N 3 I H 6CH10H 5 H Cl 0 4IH H CI OH H HoI I OH 3 2 H OH Figure 76 aDGlucopyranose H BDGlucopyranose Biochemistry 440 Fall 2008 Oxidative Phosphorylation 1 Biological Redox Reactions Reading assignment Chapter 134 p 512521 Oxidative Phosphorylation I Energy balances I Reduction potential biological redox reactions I Electron carriers I The Electron Transport Chain I Elucidation of the Electron Transport Chain I ATP synthase Glyceraldehyde 3 I he sphate Balanced equations for glucose and a fatty acid oxidation pathways I Glucose 2NAD ZADP 2Pi gt 2 pyruvate ZNADH 2H 2ATP 21120 I Pyruvate NADt CoA gt acetleoA CO2 NADH Ht Acetyl CoA 3NAD FAD GDP Pi 21120 gt 2co2 3NADH 3H FADH2 GTP CoA I Palmitoyl COA 7CoA 7FAD 7NAD 71120 gt 8 acetyl CoA 7FADll2 7NADH 7H Lecture 24 Biochemistry 440 Fall 2008 Lecture 24 Balance sheet Glucose oxidation Balance sheet Palmitate oxidation Electromotive force AE RBdUCtIOn pOtentlal E I Electrons are spontaneously donated from species with low I demon 8H1 donors to Spaces Wlth hlgher demon a mlty I One way to consider the potential energy of electrons acceptors 7gt favorable process I Reduced donor Oxidized acceptor I A measure of the tendency of a chemical species to be reduced or oxidize neg I Oxidized donor 3 Reduced acceptor l Low electron af nity gt gt I Driving force behind the electron ow Electromotive force AE AB is the difference inreduction potential E between electron I ngh electron af th gt gt acceptors and donors 39 AE Eacccpm Edam I AB is related to AG free energy change of a redox reaction gt How to quantify AG inF AE n number 0 F Faraday s c f electrons transferred onstant 965 k Volt1 mol 1 8 Biochemistry 440 Fall 2008 Lecture 24 Measurement Of reduction potentials g Reduction potential compared to a reference cell Redox reaction XCDlt Bmd lt7gt de BCDlt XBX electron acceptor BM1 electron donor Written as two half reactions reductions e39 lt7gtXM1 BBX nHt ne7 ltgt BMl 1 atquot H2 salt bridge XBX and de as Well as BMI and Bax are redox couples or redox pairs voltmeter quot Standard conditions 1 M everything Compared to a reference ZHt 2e lt gt H2 arbitrarily set at zero If E0 V negative X xt HW e39 ltquotgt Xred If E0 V positive B XnHner ltWgt Bad g The biochemical standard state pH 70 a some Standard Reduction Potentials 7 7 Acceptors Donors E 0 V EU 1 M everything except 1 X 10 7 M Ht 12 02 H20 0 82 FAD NOTE The reference is the same FADH2 0 03 l NADt NADH 0 32 ocketoglutarate isocitrate 0 38 For a given redox couple eg NADVNADH Negative standard reduction potential E gt Negative standard reduction low electron affinity potential for the redox couple XEX de Positive standard reduction potential E high electron af nity salt bridge Biochemistry 440 Fall 2008 Lecture 24 The difference in standard reduction otential AE i0 is used to analyze AG O The actual reduction potential E depends on concentrations of e d0n0rs and acceptors Whole reaction Whole redox reaction Half reactions NAD 1soc1trate ltgt NADH H ocketoglutarate CO2 XOX Brad ltgt Xred BOX XOX nH ner ltgt xred acceptor Two half reactions AE 0 V Box quotH quotequot ltquotgt Bred donor aketoglutarate CO2 2H 2e ltgt isocitrate 038 E d E f llAJr 28 ZI IJr ltgt I IJr gt acceptor an donor rom X Nernst E uation E E 0 M1 L Which is the electron acceptor acceptor 71F Xred What are AE 0 and AG 0 for this reaction 2303RT B 0 ox 0 0 0 AE E acceptor E donor doneV E n log Bred I0 I0 AG nF Eacceptor Edonor nis the number of electrons gt F is Faraday s constant 965 k volt 1 rnol 1 R is the gas constant 8315IK l mol 1 T is absolute temperature AGznFAE NAD and NADP are soluble FAD and FMN are enzyme electron carriers bound electron carriers isoalloxazine ring quot A H O I O I o H CH H if H Hi H H 3 Nrknn H UNIKNH quot395 CH3 NfKNH K 2 K a IN to N NAG CH 1NI 0 I quotH2I I or I I lquot I I I 1 R H 0 CH2 o N 2H quotll ll FMN HCOH FADH FMNH FADH2FMNHZ I H H R A side R B side MID semiquinone muyreduced o p o H H NADH HcloH l I OH OH NH reduced THZ 2 FAD o I N 0P 0 lt f Adenme 0 Q I N N o NH O CH o 2 2 O FI 0 N N H H 0 lt J NAD H H N N ca 0 OXIdIzed OH OH In NADPquot this hydroxyl group N H is esteri ed with phosphate Flavin adenine dinucleutide FAD and flavin mononucleotide FMN Flgure 132421 15 beam 17 Flgure 1327 16 Biochemistry 440 Fall 2008 Electron carriers Ubiquinone Q H3 Cytochromes Electron carriers S Cys l Cytochromes m cm Cys I CH3CH CH3 I CH3 CH2CH2C00 CH3 CH 2CH2COO Heme C I in type cytochromes CH3 CHCH2 I I H Wall CH CH3 CH3 CH3 CH3 CH3 CHzCH1COO Heme A in Hyue cyrochromes CHO CH2CH2C0039 F1 gure 193 18 ongo GHQ CHC CH2aH Ubiquanpgu Q CHao CH3 fully oxidized I o l H39 e I 039 CHgO R O Semiquinone radical QH39 case OH I on Mr H p H CH30 R Ubiquinnl 2112 fully reduced CHso CH CH F1 gure 192 17 Electron carr1ers Iron Sulfur Protelns la H c B Cys t cys cys Cys gymRag Cys Cy a E39 339 39 w l 9 Cy s 4M Cy CVS CVS Cys O Q Cys Protein Figure 195a Lehningerl rinzip IEmmzminry mm Edm39an mosw H mm m umpmvy Reduction potentials vary from 065 V to 045 V depending on microenvironment in the protein Fig 19 5 19 Lecture 24 Protein Purification amp Characterizationquot Protein Purification 1 Sources of proteins I Protein purification Natural II Protein characterization Recombinant ASSIGNED READING Chap 3 Section 33 Chap 9 p 312317 amp Sect 93 2 Extract Pmtems Web Resources Lehninger textbook site Chapter 3 39 H m gemze 39quot lyse Technique animationsquot 39 Protect The protein from BIOC440Lecture 8 Protein Purification 3 Separate fractionate proteins acc39d to different PI PH Where NET charge on protein 0 properties Exploit differential solubility a protein39s solubility depends on salt I and pH If negatlve gt pOSI39i39Ive pI 7 As ionic strength increases solubility Most proteins have MINIMAL solubility at pH pI Protein pI is pH where charge on protein 6 Fraction of soluble protein varsus pH imam If negative lt positive pI 7 Protein quotBquot s u s E a 2 4 3 Can provide modest purification use in early stages of protocol Protein Purification Column chromatography Can separate mixtures of proteins acc39d to properties wch as Mixture is applied to a column containing a solid support resin followed by buffer Separated proteins flow off column to be detected amp collected fraction collectorquot5 Chromatography based on SIZE Size exclusionquot or gel filtrationquot like a molecular sieve resin beads has tiny holes 1L 3 Purous R Jr polymer beads b z r h 3 v l l l L l Protein mixture isadded l 5 o l tomlumn mntaining r 393 r crossrlinked polymer 39 Protein molecules separate ysize larger molamles pass more freelyappearing inthe earlieriraztions Fig 113456 Chromatography based on CHARGE Ion exchangequot chromatography resin beads is charged either ve or ve I nltpuxitwulravge in buffer w low ionic strength proteins with opposite charge will bind stick to resin 51 as buffer w increasing NaCl is E flowed onto column the ions compete w protein to bind to resin Ie the ion bound to resin EXCHANGES lst if protein then Na or Cl39 Chromatography based on Specific BINDING Affinity Chromatographyquot resin has a specific chemical mg group to which YOUR protein W will selectively bind ligand Ligand 53 other proteins with low or no AFFINITY for ligand will flow through column a bound protein is eluted 1 chased off column amp collected v gt Unwanted proteins I I l rotein otinterest are washed through 5 i quot Iseluted by llgand column 13 45 3 4 S a 7 a solution 4 3 Mixture of proteins 3 ligandspetiiiclnr protein uiinruest 39 Chromatography based on Specific BINDING 1 Can take advantage of known intrinsic binding function of a protein Ligand can be 1 small molecule 2 macromolecule 2 Can engineer specific binding functionality onto an end of your protein by recombinant DNA technology 1 His tagquot 2 Protein fusion Visualizing Proteins Polyacrylamide Electrophoresis gt proteins migrate through electric field applied to a polyacrylamide gel separate acc39d to charge amp mass 1 can be visualized using a dye that sandm binds to all proteins Coomassie bluequot WWH silver stainquot etc salon uquot Iquot O u u quot39 quot each band corresponds to a speCIfIc nyoooA 3 protein I 21500 1440039 I 10 Electrophoresis based on MW SDSPAGE 1 2 Run under denaturing conditions 1 Proteins are mixed with SDS detergent amp ME reducing agentcontaining buffer and boiled G in 2004mm Unknown protein 2 SDScoated proteins migrate based solely on their MW Relative migration Electrophoresis based on MW SDSPAGE What can you learn about a protein 1 How PURE is my protein My standards 97400 66100 45000 31000 3 21500 14400 How many subunits are in my protein BIOC440Lecture 8 Biochemistry 440 Fall 2008 Oxidative Phosphorylation 11 s The Electron Transport Chain Reading assignment Chapter 191 p 707722 The electron transport chain g schematic overview lntermembrane space P side E 1 702 2H H20 NADHH NAD Succinate Fumarate Matrix N side Lecture 25 2 NADH transport into the matrix Malateaspartate shuttle inmrmomInam HF Matrix spat NAD NADt 39quot Malate 313W NADH CHIN t M NADH oxaloacetate oxaloacetate O Glutamate Glutamate WM quot 39 MHWHH 7 mlirluglulman xketo lutarate g xketoglutarate t aspartate aspartate mum Figure 19 29 NADH transport into the matrix Glycerol 3phosphate shuttle Intermembrane Glycolysis space F side NAD NADH H cytosoli glycerol 3phosphate dehydrogenase C39HZOH c 0 Glycerol 3 Dihydroxyacetone l phosphate phosphate CH2 Matrix N side Lecture 25 Biochemistry 440 Fall 2008 Lecture 25 Entry of electrons from Boxidation and citric acicl cycle lntermembrane Glycerol Glycerol space P side 3 th 5Phate 3phosphate dehydrogenase ikytosolic ETFQ oxidoreduttase Succinate Fumarate ETF NAD NADH H ACYI39COA dehydrogenase Matrix N side Fatty acyI COA EnoyICoA a The Oxidation of NADH NADH Ht 12 02 gt NAD H20 AE O 114 volts E 0accept0r E 0d0n0r AG 0 2 nFAE 0 AG 0 2 220 k Inol Enough to make 7 mole ATP per mole NADH The actual yield is only 25 mole ATP Lecture 25 E 0 Cytochrome c oxido Cyt C 3 reductase Complex IV C gt Electron Transport Chain ETC 0l4 Complex I F NADH Fes dehydrogenase Complex H FA Succinate Fes Complex III dehydrogenase Ubiquinone Cytochrome Cyt b cl Fes oxidase Cyt a 213 Cu Complex 1 REESE mm quot NADH dehydrogenase Oxidizes NADH and reduces CoenzymeQ Matrix NADH w Series of Fe S centers Figure 1 99 Lzhnirlgvi39rindples ulEiodlemisny Fihh Edition mow n r v n and Company Lecture 25 Biochemistry 440 Fall 2008 Complex II Succinate dehydrogenase Complex II oxidizes succinate and reduces CoenzymeQ lntermernbrane space F side Phosphatidylethanolamine mm m w i H l l l C l ifquot ll 9H1 M l Mi quot39f H 39Uiaiquino39ne l Matrix 139 gt39 TV Fe S centers 1 y lquot s39de Hemeb I 1 Figure 1910 The complex III mediated Q cycle Oxidation of Oxidation of first QHZ second QH lnlermembrune 5 space P side Matrix N side QH 39Q 2H cytc mxidized gt QH2 cytc1 oxidized gt QHZ 2H Q cytc reduced 390 2H cyt reduced Net equation QH2 2cyt c1 oxidized ZHN gt 0 2ytcI reduced 4H Complex IV reduces oxygen to water on the matrix side Complex IV Cytochrome oxidase 4Cyl 4H Intermembrane space F side 02 Matrix N side 4 substrate 4Hquot Pumped Complexes III and IV might associate in membrane bound super complex Respirasome g I indiumiskry rim Edirlon any e um uprian es an x 2003 WHFyeeman and amp Lecture 25 Lecture 25 Biochemistry 440 Fall 2008 Summary of electron and proton i ow through the ETC Inlermembrane space 9 side 2H go H20 NAD NADH H Matrix u side Figure 1916 zhninguPrinriplex uIBiuchamixxry mm mm zouaw n Fveenun and om nny Lecture 25 The free energy released by electron transport is conserved as a proton gradient and captured by ATP synthase UV absorption spectra Oxidized NAD Absorbance o b O l l i l l 220 240 260 280 300 320 340 360 Wavelength nm b Lecture 25 Reduced NADH 380 Electron Transport Chain Elucidation of the u The order of the electron transport chain Dif cult to study because function of ETC depends on maintaining the structure of the membrane gt Use of inhibitors in intact mitochondria gt lnfer ow of electrons from spectra Answer two questions Lecture 25 Lecture 25 Lecture 25 i Experimental Plan using Inhibitors of the ETC Treat mitochondria with antimycin A I Find that NADH Q and cyt b are reduced Cyt c1 cyt c cyt a and cyt a3 are oxidized What electron carriers are upstream of the block the reduced carriers or the oxidized carriers Why Biochemistry 440 Fall 2008 04 Complex I FM NADH dehydrogenaseFeS Csomplex II FA uccmate F S dehydrogeuase e G Electron Transport Chain Complex 111 Ubiquinone Cytochrome c oxidov Cyt C reducIAse Complex IV c b c Fes ernfhrnme y 139 oxidase Cyl a 213 C Experimental Plan using Inhibitors of the ETC Treat mitochondria with arnytal Find that NADH is reduced Q cyt b cyt c1 cyt c cyt a and cyt a3 are oxidized Lecture 25 Lecture 25 i Inhibitors of the ETC Complexl gt Q gt 111 gt cyt c gt IV Lecture 25 Redox Reactions Redogtlt is n m i A A h types to substance combining with oxygen as nappens wnen an exarnpies as Corrosion and The terrn oxidation originaHy referred r a camp re iog bums We often refer to tnese two iron bar ruSB O combusuon 39 a process tnat is accompanied by a reduction H i tne mass of tne ore These two terrns nave broader meanings now in 5H Oxidationrreduction reactions an excnange of Thatis tne key to understanding redox reactions in excnange reduction reaction LE0 LE0 Loss of Eleclmns Is le a nn the Lion Says GER Gem of Elev mns ls Reductlnn GER Formal oxidation numbers gt A simple method to be used for BIOC440 carbon centric different from the textbook A carbon gets 1 for each bond to hydrogen 0 for each bond to carbon 1 for m bond to O N or S gt Then sum up example Acetone H O H HCla lH the carbonylcarbon has 2 H H the methylecarbons each have 3 pH Why should we carequot I Biochemistry is pHdependent II How buffers work A General principles B Practical considerations C Buffers in biology ASSIGNED READING Chap 2 Section 22 amp 23 For a more thorough description of blood buffering see Web links listed on BIOC440 Schedule page REVIEW amp TEST yourself online see Web link listed on BIOC440 Schedule page HOMEWORK PROBLEM SET 1 BIOC 440 Lecture 2 1 Biochemical processes amp Biological Systems amp pH reactions are pHdependent Cells amp organisms maintain constant specific pH value that is optimal for function Example blood pH 74 IF blood pH lt 68 or gt 78 l cvcem maximum netwitv Buffers in Biochemistry in viva amp in vitro Gen39l Def39n Practical Def39n z x z r o How Buffers Work Henderson Hasselbalch Eg39n so much info in so little time How Buffers Work 2 Equilibrio Involved HAc 2 H Ac H OHquot 2 H20 3 choo 7 39 CH3COOH CH3COO 6 pH 576 5 39 Buffering PH 4 pHpka4JG szJG 3 2 CH3C00H 39 1 n I I I I I I I I 01 02 03 04 05 06 07 08 09 10 OHquot added equivalents 0 50 100 Percent titrated Hg 2 16 5 Shape of titration curve is same for all weak acids Midpoint 1 3 e of titration 12 e l Bufferin NH 9 m L 2 8 H303 IHPOi T786 7 Phosphate pKa 476 T5275 5 39 Acetate 4 CH3COOH CHZCOO 33975 3 2 I I I I I I I I I I 0 01 02 03 04 05 06 07 08 09 10 OH added equivalents 0 50 100 Percent titrated l g2 17 How Buffers Work The right buffer for the job maximum buffering capacity buffering range buffer concentration amp capacity Biology39s buffers Physiological pH 74 blood In vivo buffering systems Carbonic acidBicarbonate system multiple equilibria K K K H30 HCOs39zlt HZCOS Ham 2 ZHZO com 716029 What39s happening in the body The blood buffering system simplified COZ is produced by cellular respiration HHco 3 Overall equilibrium in sol39n COZ is converted to bicarbonate rearliaul K by carbonic anhydrase an enzyme Hm H HCOS39 2 COM It 39 LOWER H in quot quot resu S In P bloodmcapillarles quotMIan Kh where K Ka Kh respiring iSSles 51t 39mi fiii quot l n H20 H10 COZ is exhaled in lungs ixmmmrn N 1 I cw A modified Henderson Z xmm m unmi I xclltgsxlx ntlii mamaquot Hasselbalch Eqn nrespirlng issues Gas phase ll lungairspace cozlgl 39 39l39 3909 W31T cnrlxmii Imh lmsx 202HgO gtIIC H 1 9 33C39 a 72AI2lt11CO 11 r 390 31 9 cmimnwunhydmsu C02d lnlimgs 61 log HC03 3903 Him 1 x O lurlmn xlinxirlv Ituvos liicurlmlnuu rimrs w El f i quot 10 The blood buffering system simplified When things go wrong WW3 Under normal condi ons Alkalosis a condition of excess base in body fluids readioM Acidosis a condition of excess acid in body fluids Aqueousphase Him bl d39 39ll 39 reaction 00 Intapl il IES H20 H10 E col hyperventilation Wequot Du severe diarrhea Gas phase l rmg exemlse39 excess loss of bicarbonate lung air space W extreme prolonged exercise 559 2 20 HCO lactic acid build up pH 61 log C O3T prolonged vomiting 2 kidneys retain H603 Q Normal blood pH is 74 Is 60201 gt lt HCO339 39 ex lr eme Pne l decreased breathing 11 12 REVIEW How 1390 calculate an EQUIVALENT for a buffer General form of NET Rx a HA OHquot 2 Aquot H20 6 pH 576 5 Bulfering Pl39l vegion 4 pH 376 pHp a476 2 I I l l x l l l 0 0 02 03 01305 07 03 09 10 OH39added equivalents 50 100 Percent tilvaled Fig 2415 BIOC440Lecture2 13 Biochemistry 440 Fall 2008 Lecture 19 Regulation of Glycolysis i and Gluconfogenesis Reading assignment Chapter 153 p 582 590 591 594 optional Steady state and Flux Consider the reaction I A gt I gt P I When V1 V2 then I is constant I If V1 V2 gtgt 0 then ux is fast I If V1 V2 gt 0 then ux is slow Lecture 19 Biochemistry 440 Fall 2008 Lecture 19 39 392 How IS ux regulated Ways to regulate enzymes w a Extracellular 7 si nal l Change in substrate concentration S 39 I 9m rnost physiological reactions quot 39 52230133337phospmmn ltlt K Transmfrc39toari EZZTa39I39 iimin 9 rn Transcription nf a Enzyme binds gt r r specn zgenem kinase phusphanase ligand I w allostenc BUT h siolo ical S onl va about 2fold DNAWW W p y g I y ry Nucllus J 39 g V 7 mnNAdegradamn 39 f gt W H Product Enzyme binds l ql 1r substrate gt a 3 Ei39 39 il a39iifj iue Regulation of enzymes 33 aaski m 9 mRNA translaxiun on ribosome r idrgme Lecture 19 3 Lecture 19 4 Biochemistry 440 Fall 2008 Lecture 19 Ways to regulate enzymes l Change amount of enzymes present gt level of synthesis transcription translation and degradation proteolysis gt sequestration into compartments In uence enzyme activity Lecture 19 i Allosteric Regulation T state I R state I Allosteric effectors shift this equilibrium Inhibitor shifts the equilibrium towards Activator shifts the equilibrium towards Lecture 19 Biochemistry 440 Fall 2008 Covalent posttranslational modi cations Phosphorylation ATP ADP V Inactive enzyme ctive enzyme Lecture 19 1 Lecture 19 Glycolysis Gluconaogonesis ATP Glucose pI Glycoly81s and mmf yummham lucose gluconeogeneas ADP efhospham H20 ATP Fructose pi phospho 639phospha e fructose iruztokinase1 F t 16 bisphosphatase1 run 052 ADP 16bisphusphate H20 Dihydroxyacetone Dihydroxyacetone hosphate phosphate 2 Glyceraldehyde 3phosphate 2 P 2 P 2 NADt 2 NAD 2 NADH 2 H 2 NADH H 2 13Bisphosphoglycerate 1 2 GDP 2 ADP 2 ADP 2 ADP Phosphot llpyruvate PEP arm 2 ATP 2 ATP quotmm mm m Oxalouei 2 3Phosphoglycerake 2 ATP 2 A 2 Pyruvate pyruvate carl 2 ATP 2 2Phosphoglycerate Biochemistry 440 Fall 2008 Lecture 19 Regulation of Glycolysis PhosEhofructokinase PFK l g PFKl aCtIVIty VS39 F6P m Phosphofructokinase The curves represent I Inhibited by ATP citrate the activity of phospho Activated by ADP AMP Fructose 26bisP fructokmase P3191 g under two condltlons gt ATP AMPADP Low ATP J I High ATP EL it Fructose 6 ATP Fructose16 ADP r 7 t 7 M phosphate bisphosphate l l l citate iructose 26 bisphosphate Fructose 6phosphate b 10 Lecture 19 Biochemistry 440 Fall 2008 Lecture 19 Regulation of Glycolysis Hexokinase and pyruvate kinase Fructose metabolism revisited Hexokinase muscle 39 Inhibited by Glucose 639P In liver fructose catabolism bypasses the first four steps of glycolysis I Pyruvate kinase The major regulated step of glycolysis is bypassed Inhibited by ATP acetylCOA long chain fatty Step 3 phosphofructokmase PFK1 ac1ds alanlne I Activated by F 16BP What would be the effect of ingesting fructose on the levels of pyruvate Lecture 19 11 Lecture 19 12 Biochemistry 440 Fall 2008 Lecture 19 AMP ADP ATP Citrate Lecture 19 Glycoly sis Phosphofruc rokinase PFKl F26BP Fructose 6phosphate Fructose 16bisphosphat Regulationiof PFK l and FBPase1 Gluconeogenesis ANIP F26BP Fructose 1 6 Bisphospha rase FBPase ll O P 0 CH2 CHZOH I o o H HO H H 0 rgt o OH H 0 Fructose 26bisphosphate Biochemistry 440 Fall 2008 Lecture 19 100 gtF 80 LH O Q E 60 E E s 40 CG 7 m h 20 D4 0 Figure 1516 0 1 1 1 1 1 l 1 l 1 1 1 005 01 02 04 07 10 20 40 Fructose 6 phosphate mM a FBPasel activity of Vmax Figure 1516 100 80 60 40 20 F2 BP 1 1 1 0 50 100 Fructose 16 bisphosphate MM b Biochemistry 440 Fall 2008 Lecture 19 Synthesis of Fructose 26 bisphosphate i Regulation of F 26BF levels Fructose 6 phosphate S I Phosphofr39uc rokinaseZ FFUClose 216 b39sphosphamse I What effect would phosphorylation of FBPase Z PFK Z have on F 26BP levels What effect would this have on glycolysis and gluconeogenesis Fructose 26 bisphosphate These enzymes are on a single polypeptide chain and regulated by phosphorylation Lecture 19 Lecture 19 Biochemistry 440 Fall 2008 Lecture 19 Regulation of Pyruvate kinase and i PXruvate carbroxylase GIYCOIYSiS Gluconeogenesis 139 phosphoenolpyruvate PEP carboxy W kinase ATE1C A oxaloacetate ace 0 long chain fatty aUd pyruvate alanine Pyruvate F 16BP carboxylaSe Lecture 19 Regulation of i Gluconeogenesis and Glycolysis Lecture 19 I What would be the metabolic effect of increased levels of acetyl CoA Biochemistry 440 Fall 2008 Lecture 16 Thermodynamics 8 Biochemical energetics AG the change 1n free energy Thermodynamics Free energy change of a reaction AG Standard free energy change AGO Relationship of AG and AGO Bioenergetics Coupling of reactions ATP Activation barrier transition state l AGtm M h Products 8 Reaction coordinate A gt B Free energy G Reading assignment Chapter 131 amp 133 box 13 1 optional 132 for review only Lecture 16 1 Lecture 16 i What is AG g What is AG Equation 1 AG is the change in free energy AGszichiRT1ncgng AillBil Equation 2 I Consider the reaction A B ltgt C D Ci HQ gt Initial conditions Ai l3i ltgt Ci Di AG RTln A RTaneq gt At equilibrium A6 Be ltgt C6 De 1 l R is the gas constant T is the temperature in degrees Kelvin Lecture 16 3 Lecture 16 4 Biochemistry 440 Fall 2008 y Age is the Change in Standard free energy AG0 is the change in standard free energy I At standard state the concentrations of reactants 39 SOIVII gEquatlon 3 SW95 and products are Equahon 4 Lecture 16 AG 2 RT1n Km AGquot 2 2303RT log Km 39 AGO is the Change in free energy going from 2 303RTis approx 5 kImole at 25 C I Substituting into Equation 2 gives I In biology we use AG39O Equation 3 Standard state with additional assumptions 1 1 pH 70 AG 2 RT lnm RT 1n Keq 55 M H20 1mM Mg2 K eq makes the same assumptions Lecture 16 5 Lecture 16 l The relationship of AG to AC Considerthe reaction A ltgt B I Substituting equation 4 into equation 2 gives Equation 5 AG AGO RTI D1 n A B l 1 Given the reaction What is Q I Change to log base 10 to give C D A lt gtB Wh t AGo Equation 6 AG 2 AG 0 23RTlog If A 0 1 M Wha ls G A i at is A and B 1 M In which direction will And K39eq 100 the reaction proceed I AG gt 0 gt I AG lt 0 gt Lecture 16 7 Lecture 16 Biochemistry 440 Fall 2008 Lecture 16 I I What can we read from K AG 0 and In uence of Q on AG eq Q 7 From table 153 I Reaction AG 2 0 Ma mmquot mm equ39illflmum A5 MAflu Enzyme K m Liver Heart in vivo Idmol in heart Hexukinase 1 x103 2 gtlt1tr2 a x10 2 No 17 27 AG 5 kJm011OgK 5 kJm01QgQ PFK1 10x103 9x10 2 3gtlt1Ir9I No 14 23 sq Aldolase 10 x 10quot 12 x 1V5 9 x 10 6 yes 24 60 AG and Keq are characteristic constants for a reaction Q is Variable gt in uence on sign and magnitude of AG Lecture 16 9 I I Reaction coupling through formation Overall AG 0 and K eq of coupled Of a COHIHIOI I intermediate reactlons kI mol Citrate ltgt isocitrate 5 AG 1 Coupling of an unfavorable reaction with a favorable reaction through a Isocitrate if NAD ltgt Pketoglutarate NADH H 02 21 AG 2 common intermediate such that the overall reaction is favorable Citrate NAD ltgt diketoglutarate NADH H co2 k mol Citrate lt gt isoc1trate 5 AG 1 lsocitrate NAD lt gt diketoglutarate NADH H CO2 21 AG 2 Citrate d Citric 1509mm 5 a commonlme me late 39gt Citrate NADt ltgt diketoglutarate NADH H co2 16 AG verall acid Isomtrate constantly consumed by the favorable cycle followup reaction 06 ket0 gt small value for Q l The overall AG of a coupled series of reactions glutarate l The overall K e 1 Metabolic reactions Within one pathways can be coupled They need to be considered Within their metabolic context Lecture 16 11 Lecture 16 12 Biochemistry 440 Fall 2008 ATPcoupled reactions Lecture 16 AG 0 kIrnol ATPHZO lt gt Pi ADP 0 5 PiGlucoselt gtGlucose 6PHZO 13 8 ATPGlucoselt gtGlucose 6PADP 167 I ATP is the most widely used i energy currency of the cell Adenosine triphosphate ATP P P adenosine P adenosine Lecture 16 Lecture 16 ATPcoupled reactions in viva Direct cou ling by an enzyme ATP Glucose ltgt ADP Glucose6P Direct phosphorylgroup transfer no Pi intermediate bond energy preserved Lecture 16 ATPhydrolysis Hydrolysis ATP H20 gt ADP Pi AG 305 kImol Phosphoryl group has high transfer potential Hydrolysis of ATP is favored because I ATP is kinetically stable gt high activation energy for hydrolysis Lecture 16 Biochemistry 440 Fall 2008 5 Lecture 16 O O O i o rl liib1 o 1 fwo O 0 117027 0 ATPquot i1 hydrolysis with relief of charge repulsion l u u 39O P OH iio r r O i v P 0 7 O ADPZ Aresonance stabilization i wmzauon 5 3 3 l n 570ff70395 H7 H4r 0713707137 Rib Adenine C 0 0 ADP3 ATP H20 gt ADP P H r AGm 305 kJmol Figure 1311 17 ATP does not have the hi hest transfer potential Lecture 16 Phosphoenolpyruvate Creatine phosphate ATP Glucose 6phosphate AG39O 619 k mol 430 kJ mol 305 k mol 138 kJ mol energy by Lecture 16 ATP provides group transfers Figure 1318 a Written as a onestep reaction 300 C00 1 H3NCIH ATP ADP Pi H3N CH CIH2 NH3 CH2 CH2 CIHz C C 0 O 0 2 Glutamate Glutamine ATP NH3 ADP 00 D 3 quotIquot P 1 2 C 0 O O P 0 O Enzymebound glutamyl phosphate b Actual twostep reaction Lecture 16 Biochemistry 440 Fall 2008 Lecture 20 Glycogen Metabolism i and its regulation Reading assignment Chapter 154155 p 594 604 605609 optional i Glycogen Storage form short term of glucose in vertebrates and many microbes starch in plants Mainly formed in liver and muscle to provide a quick source of energy Glycogen in liver is a reservoir for other tissues during fast brain muscles polymer of a D glucose Lecture 20 Biochemistry 440 Fall 2008 Lecture 20 Structure of Glycogen Structure of Glycogen 1 10 of the linkages are oc1 gt 6 CHZOH CHZOH OH OH o o 2 O H OH 1 4 OH OH OH l O I OH OH OH OH CH CHZOH O Most linkages in glycogen are oc1 gt 4 OH OH OH Lecture 20 Lecture 20 OH 3 4 Biochemistry 440 Fall 2008 Lecture 20 Glycogen Glycogen CHZOH cuzon o o m CHXOH H o H H H H H H H H HO on H 0 mm H 0 OH H amunkage H OH H oH H on Reducing 0 and N oureduc g m ends cHoH CH OH CHI H10 H 0 H H 0 H H 0 H u 0 H O H H H H H OH H OH H OH H OH H Ho 0 o o 0 0H H OH H on H oH H on oH Branch oint 34 Reducing p linkage a Brapch end quotquot paint Lecture 2U Lecture 2n Biochemistry 440 Fall 2008 Lecture 20 Glycogen Nonzentzlfing 39 breakdown 0 G1 CO en breakdown phosphorolys1s CH20H CH20H CHon 0 o 0 OH OH OH O O O O O O O 0 00 OH O O Glucose 1phosphate OH OH OH molecules CH20H CH20P03239 o 0 OH 6 OH gt gt pyruvate Q Glucose OH OPO32 OH OH glycolysis Unbranched OH OH gure 1526 ongt4 polymer W77 merfajfucose 1P Glucose 6P 8 Biochemistry 440 Fall 2008 Lecture 20 i Glycogen Breakdown i Glycogen Debranching Enzyme I What would be the product if the CHZOH CHQOH CHZOH OL1 gt4 bonds were broken by hydrolysis OH O OH O OH O ie if the bonds were attacked by H20 instead of Pi OH OH 0H 0 x1 gt 6 I Would this be as effective a use of glycogen CHZOH CHQOH CH2 as an energy source OH O OH O OH O OH OH OH OH Lecture 20 Lecture 20 Biochemistry 440 Fall 2008 Lecture 20 Glycogen breakdown Debranching enzyme i summar Qf reactions I Phosphorylase can only cleave residues I PhosphordySlS Ofoc1quotgt4b0nds that are four or more away from a I Conversion of glucose lphosphate branch to glucose 6phosphate I oc16 glucosidase can only hydrolyze a branch of one residue I gt transferase activity is essential I Hydrolysis of 0c1gt6 bond component I Trisaccharide transfer I further phosphorolysis of 0c1gt4 bonds Lecture 20 Lecture 20 12 Biochemistry 440 Fall 2008 Lecture 20 g Thermodynamics of Glycogen Metabolism Glycogenn1 Pi gt Glycogen11 G 1phosphate l AG 0 of the hosphorylase reaction is 31 k moi In the cell the ratio of G1P Pi ranges from 130 to 1100 so this step is exergonic Q lt Keq gt Glycogen synthesis is NOT the reverse of glycogen breakdown Lecture 20 Comparison of i Breakdown and Synthesis Breakdown Glycogenm1 Pi gt glycogenn Glucose 1phosphate Synthesis Glucose 1phosphate UTP gt glucoseUDP PPi PPi H20 gt 2 Pi Glycogenn UDPglucose gt glycogenm1 UDP Lecture 20 Biochemistry 440 Fall 2008 Lecture 20 Glycogen synthesis i Synthesis of UDPglucose I UDPglucose is made I GllJ UTP gt glucose UDP PR I Glucose is transferred from UDPglucose 39 to glycogen I AG O 0 kmol gt making ochgt4 linkages PP H O gt 2P I ocl gt6 linkages are introduced I 1 2 1 gt making branches 39 I AG O 25 kImol Lecture 20 Lecture 20 Biochemistry 440 Fall 2008 Lecture 20 S nthesis of 0c 1gt4 linka es G1 CO en Branchin y 7 g y g g CH20H CH20H CHZOH O O O I 3 OH OH OH 0 1 OH O UDP OH R quot o o o o OQOJOLOJOLOJOLOQOQ Glggen OH OH OH Nonrg mg quot194 glycogenbranching UDP glucose Glycogenn 1quot i mmmm CHon CHZOH CHZOH Nunreelting HQOQDQOD Glyc O O 0 core OH OH OH OH R OH OH OH GI co en Branchin Enz me Lecture 20 Glycogenrprl bcturfigufe 15 31 y g g y 17 18 Biochemistry 440 Fall 2008 Lecture 20 Glycogen Storage Diseases i Regulation of Glycogen Metabolism I 9 different types I Phosphorylase is activated by I Vll Lack PFK in muscle phosphorylatlon gt regulated by hormonal control I I lack of glucose 6 phosphatase affects gluconeogenesis and glycogen storage I Glycogen synthase is Mactivated by I III lack of debranching enzyme phosphorylation I IV lack of branching enzyme Lecture 20 Lecture 20 10 Biochemistry 440 Fall 2008 Lecture 20 Ser14 Ser14 side 1 Cf side Glycogen phosphorylase 1n liver chain CH2 CH2 chain Phosphorylase b GP less active 390 393 CH2 CH2 2 Glucose 2P 2ATP I phosphorylase a Phospharylase a phosphorylase a phosphorylase b Glc Glc phosphatase kinase PM 2H20 2ADP I t lH H 0 0 CH2 CH2 CH2 CH2 CH2 CH2 zpi phosphorylase a Phasph39zrylase a L Phosph39nrylase b active Gk Glc phtfsphclnrylase a Gquot Gk Figure 1534 PP1 Flgure 1536 less actlve 22 11 Biochemistry 440 Fall 2008 Lecture 20 a Glycogen 0 Storage form shortterm of glucose in vertebrates cogen Metabolism and many microbes starch in plants i and regulation 0 Mainly formed in liver and muscle to provide a quot quick source of energy 0 Glycogen in liver is a reservoir for other tissues during fast brain muscles Reading assignment Chapter 154455 p 594604 605609 optional 0 polymer of ocD glucose Lecture 2n i Structure of Glycogen a Structure of Glycogen l 110 of the linkages are oc1 gt 6 CHQOH CHQOH OH OH O O 2 O OH 1 4 OH OH OH O I OH OH OH OH OH CHQOH Most linkages in glycogen are 0 l gt 4 OH OH OH Lecture 2n Lecture 2n OH 3 4 Biochemistry 440 Fall 2008 Lecture 20 Glycogen Glycogen CH20H CHEOH cuzou w 3 a H 0 H H o H H 0 H l g H H Nonrgggcmg 0 H0 H 0 0 H o 0 H all a linkage o o f H OH H OH H OH Reducing 0 end Nonreducing ends CH20H engoH CHz CHIOH CH20H H 0 H H 0 H H 0 H H O H H O H H H H H H OH H OH H OH H Ho 0 o 0 OH H 0 OH H OH H OH H OH H t OH H OH H OH Branch point we Reducin w linkage 0 Branch end g pomt 0 Lecture 20 Lecture 20 5 6 Glycogen quot2 i 332 breakdown G1 co en breakdown phosphorolys1s GI co en y g CHZOH CHZOH CHZOH I o O O OH OH OH 00000 ma 0 OOOOO 00 OH O O Glucose 1phosphate OH OH OH molecules I CH20H CHZOPoaz39 A O O OH gt OH gt gt gt gt pyruvate Q Glucose OH OPOGZ OH OH glycolysis F1 1526 Unbranched OH OH gm ongt4 polymer my Lmu fllcose 1P Glucose 6P 8 Biochemistry 440 Fall 2008 Lecture 2D Glycogen Breakdown I What would be the product if the OL1 gt4 bonds were broken by hydrolysis ie if the bonds were attacked by H20 instead of Pi I Would this be as effective a use of glycogen as an energy source Lecture 2D l Debranching enzyme I Phosphorylase can only cleave residues that are four or more away from a branch I oc16 glucosidase can only hydrolyze a branch of one residue gt transferase activity is essential component lecture 20 g Glycogen Debranching Enzyme CHQOH CHQOH CHQOH o O 0 OH OH OH O OH OH OH O 0L1 gt 6 CHQOH CHQOH CH2 0 O O OH OH OH OH 0 0 OH OH OH LectureZD Glycogen breakdown summag of reactions I Phosphorolysis of a1 gt4 bonds I Conversion of glucose 1 phosphate to glucose 6 phosphate I Trisaccharide transfer I Hydrolysis of a1 gt6 bond I further phosphorolysis of a1 gt4 bonds Lecture 2D Biochemistry 440 Fall 2008 I AG 0 of the phosphorylase reaction is 31 kIrnole In the cell the ratio of G1P Pi ranges from 130 to 1100 so this step is exergonic Q lt Keel gt Glycogen synthesis is NOT the reverse of glycogen breakdown Lecture 2n Thermodynamics of Glycogen Metabolism I Glycogenm1 Pi gt Glycogenn G 1 phosphate i Glycogen synthesis I UDP glucose is made I Glucose is transferred from UDP glucose to glycogen gt making och gt4 linkages I a1 gt6 linkages are introduced gt rnaking branches Lecture 2n Comparison of a Breakdown and Synthesis I Breakdown Glycogenm1 F1 gt glycogenn Glucose 1 phosphate I Synthesis I Glucose 1 phosphate UTP gt glucose UDP PP I PP H20 gt 2 P I Glycogen11 UDP glucose gt glycogenm1 UDP Lecture 2n a Synthesis of UDP glucose I G1P UTP gt glucoseUDP PPi I AG 0krnol 1311 H20 gt 2Pi AG 25 kJmol Lecture 2n Lecture 20 Biochemistry 440 Fall 2008 Synthesis of 0c1gt4 linkages CH20H CHZOH CHQOH O O O OH OH OH OH O UDP OH OH OH OH UDP glucose 1 Glycogenn CHQOH CH20H CH2OH O O O OH OH OH OH O OH OH OH Lecture 20 Glycogen 1 UDP i Glycogen Branching QQ a o 61 quot33quot quot1 4 glycogenbranching enzyme Gt H H M M 9quot quot quot til gt6 Branch point Nonreducing GI K K gt ycogen end 7 a core Figure 1531 Glycogen Branching Enzyme Lecture 20 il Glycogen Storage Diseases 9 different types VII Lack PFK in muscle I lack of glucose 6phosphatase affects gluconeogenesis and glycogen storage I III lack of debranching enzyme IV lack of branching enzyme Lecture 20 g Regulation of Glycogen Metabolism Phosphorylase is activated by phosphorylation gt regulated by hormonal control Glycogen synthase is Mactivated by phosphorylation Lecture 20 Lecture 20 Biochemistry 440 Fall 2008 Ser 0H 0H Ser side I I side chain CH2 CH2 chain Phosphorylase 1 less active 2Pi 2ATP I phosphorylase a phosphorylase b phosphause kinase w ZHZO ZADP o o CH2 CH2 Phosphorylase a active Figure 1534 Glycogen phosphorylase in liver 2 Glucose o CH2 Phosphorylase a Phosphorylase a Phosphorylase a Glc Phosphorylase b Glc Phi Ph i39y39a Glc Glc r PP39I Figure 1536 less active Lecture 20 BIOC440 Autumn Quarter Review of Units Biochemistry is a quantitative science which means that using proper units is important You should have learned about units and how to convert among them in Introductory Chemistry but you may not have used them in awhile Many of the problems you will be learning to solve in this course will require use of units so you should review and re memorize the fundamental concepts such as 1 Moles mol are NOTthe same as Molar M The rst is a count ofthe number of things you have and the latter is a concentration ie the number of things you have per unit of volume in this case molL Using these two units interchangeably isjust plain wrong and will be counted as such on exams 2 Biochemistry takes place in dilute solution ie most of the concentrations we care about are much lowerthan 1 M Therefore we tend to report concentrations in units of mM millimolar 103 M uM micromolar 1O396 M 1O393 mM nM nanomolar 1O399 M pM pico molar 1012 M Similarly volumes often tend to be small mL milliliter 103 L uM microliter 106 L 103 mL Protein Structure II Covalent structuresquot I Protein structure hierarchy II Primary structure of proteins III Information content of protein sequences ASSIGNED READING p 33 36 Sect 32 amp Sect 34 only p 92 94 amp 102 107 P 378 379 Lehninger Web tutorial use Web link listed on BIOC440 Schedule page REVIEW amp TEST yourself online use Web link listed on BIOC440 Schedule page and try Probs 1 2 5 9 11 12 HOMEWORK PROBLEM SET 2 BIOC 440 Lecture 4 1 The function of a protein can best be understood in terms of its structure FOUR levels of structural organization Primary Secondary Tertiary Quaternary Primary structure Quaternary structure gt2 u Secondar Ternary structure structure E m Th r m m p I E i i gtrlt El i l 1 U lt N Amino acid Polypeptide chain Assembled subunits residue Fig Protein Primary covalent Structure proteins are polymers of AMINO ACIDS functional proteins consist of not ALL sequences exist in nature Protein Sequence Nomenclature directionality or polarity numbering convention residue What can we learn from sequence 1 Identify potential transmembrane regions Hydropathy plots cupside Aminoterminus 7 395 w x lt a i gt Winl H l 5 Hydrophobic l Hydrophilic 3 V l l l o gt V I 3 i nside 10 50 100 150 200 250 Residue number Carboxyl terminus m Mth 5 What can we learn from sequence 2 Comparison of sequences identify consensus sequences or motifs DWDNSlLVFYWDENSTGDNQGHRKGP LIVMCIDENQSTAGCx2DELIVMFYW Bits cInwh b N How Protein Sequences are Analyzed 3 Comparison of sequences identify protein families E coli TGNRTIAVYDLGGGTFDISIIEIDEVDGEKT FEVLATNGDTHLGGEDFDSRLIHYL DEDQTILLYDLGGGTFDVSILELGDG T FEVRSTAGDNRLGGDDF DQVIIDHL B subtilis lG apl Fig 3 44 invariant positions conservative changes gaps HOW similar are aligned sequences There are many algorithms available online that will perform sequence alignments Output will include o aligned sequences 0 identity 0 similar 0 Evalue Similar sequence implies similar structure amp a w v u 1 43 m is Cytochrome c DVEKEKK IMK ESQTV EKGGKHETENQHQL FGEK TEIQAPEYSMT IDA T VQR AL IDNNLGQ Q 3 IYS HS SVV FT S s A AN l N I 39r 39l l 1 ii 4 v A A v I s F q I39TA PK s 39l39 K I u r v w H STT Y D Q w m H a u r 1 gt7 mu AKNKGIIIEGEDTLME m kmvaVQIKKEEmADLlAYLRKA l lSRAVl ADENMSD T V A LS TDD CNIVTF MIDK Ii Q M N v x l N r 1 L A G x A 139 v E 39r N K A T Q Q P Y A l l N T Q S T K N D ll l T E R Q S A Q E DQ N T E K A E l I invariant positions conservative changes hypervariable positions Similar sequences RELATED functions Example the globin family of proteins 1 Compare human myoglobin Mb w ALL protein sequences 2 Compare human myoglobin Mb w all known HUMAN protein sequences Comparison of sequences shows proteins can withstand MANY amino acid changes BUT A single change MUTATION is the cause of many human diseases 1 Sickle cell anemia 2 Cystic fibrosis 3 Breast cancer 4 MANY others BIOC 440 Lecture 4 11 Enzymes I How enzymes workquot I What catalysts do II Enzymes are biology39s catalysts IIIStrategies used by enzymes ASSIGNED READING Chap 6 Sect 61 amp 62 p 183194 HOMEWORK PROBLEM SET 5 BIOC440 Lecture 12 1 Spontaneous a Speedy ecl f P AG 0 RATE of a reaction depends on AGF height of barrier depends on 1 alignment of reacting groups 2 bond breakingforming Transition state 1 0 AG 3 s gt p a Mai gt5 5 g s 1m 4 E Ground P Standardhfree energy c ange State Ground pH 70 298K state 1Wquot Figure Reaction coordinate How to get there faster 1 2 E s gt E P Activation barrier u transition state 1 E quot E a t N AGuncat 5 Reactants A A63 a 139 H E AG Products B 27 Reaction coordinate A gt B Enzymesbiology39s catalysts 1 VERY high rate enhancements 2 Work under MILD conditions 3 HIGH reaction specificity 4 Can be REGULATED Enzyme Vocabulary You Should Know substrate active site cofactor coenzyme holoenzyme apoenzyme DUIhme Naming Enzymes General rule quotSomethingquot quotasequot where quotsomethingquot is usually quotsubstratequot or quotreactionquot EXAMPLE Classifying Enzymes quottype of reactionquot quotasequot REACTION Enzyme class Oxidationreduction Transfer of functional grp Isomerization Chain building WHAT enzymes do Uncatalyzed Z P Velocity Vunm kum kuncaf kBThexp39AGuncatRT Catalyzed EZ E 5 E P EP Velocity Vcm kca km kBThexpAGmRT Transition state l Rate enhancement kmkLlncm AGquot quotmat exPAG uncat AG cogRT cal Free energy G I l l l I I I I I I gt Figure 63 Reaction coordinate AGIFB Binding Energy affords substrate orientation substrate specificity Figure 6 4 Figure 6 22 8 Transition state stabilization amp Binding Energy a No enzyme 0 t gt quot V V 9 AG 57 gt 39 J g 39 w n S Substrate Transrtlon state 2 p metal stick bent stick broken stick quotquot b Enzyme complementary to substrate 6 a 3 1 a g uncat AGm W g 35 u Lb gt quotW quot 39 39 39 39 39 g Iquot 1 AGuncat 1A M a in ES 2 P u Reaction coordinate Figure 3 5 General MechanismsStrategies 1 Acid base catalysis acidicbasic groups acceptdonate H to stabilize an unstable species R1 r W HcOH 90 Hlcoco 3932 rlqH R2 I39dH R4 R4 Chymotrypsin ser195 Ser195 3 H0 BH 0 2 1 1 R R 7 gt o R H o H RZ NH2 Acidbase catalysis Enzymes use amino acid residues in their active site as proton donors andor proton acceptors Amino acid General acid form General base form Ac ve sifes may residues proton donor proton acceptor provude special 539quot ASP R COOH R COO39 environment that H alters the K LysArg RJEH R NHZ P R cys R SH R S R C CH R CCH HIs HN N HN N c c H H Ser R OH R O Figure 9 11 General MechanismsStrategies 2 Covalent catalysis Uncat AB gt A B slow Ca l39l ABX gtAXB gt ABX Chymotrypsin ser195 ser195 3 HQ BH I c RZ l C R o R I H H RZ NH2 General MechanismsStrategies 3 Metal ion catalysis 13 of all enzymes require metals Mefal ions in enzyme acfive sifes 39 bind amp orierrf subsfrafe 39 sfabilize charged infermediafe 39 perform reducfionoxidafion chemisfry EIOCMOLecmre 12 13 Biochemistry 440 Fall 2008 Lecture 18 Lecture 18 I Carbohydrate Metabolism I Feeder pathways for glycolysis I Gluconeogenesis I Pentose phosphate pathway Reading assignment Chapter 142 p 543546 144145 p 551563 Print out gure Gluconeogenesis from the course webp age under assignment or schedule and bring to lecture Glyceraldehyde 3 I hosphate Citric Acid Cycle H20 Biochemistry 440 Fall 2008 Lecture 18 Entry of other hexoses into glycolysis Tveha lose Lactose H O CH20H 2 o 4 4 Glycogen search H H H uramylase o H Pi phosphorylase I OH UDP galactose sucrase H o Glucose lt UDPglucose Gl 1 hos hate o ucose ATP P P mutase How2 o Sucrose H0 cnon H H Ho Glucose H H OH 6phosphate 0H H ATP F hexo D ructose kinase ATP lfruclokinase Fructose 6phosphate Fructose 1 phosphate fructose 1 phosphate aldolase Fructose 16 bis hos hate Glyceraldehyde Dihydroxyacetone P P osphate uiose triose phosphate l ATP kinase isomerase Glyceraldehyde 3phosphate Fi gu re 1 4 1 O Lelminger Principles oiBiochemisrry Fifth Edition nn w u hexokinase phosphogluw HO o H H OH H H OH H DMannose Fructose metabolism In muscle Hexokinase phosphorylates fructose Fructose 6phosphate continues through glycolysis In liver Glucokinase instead of hexokinase Glucokinase is speci c for glucose gt Instead Fructokinase Lecture 18 Biochemistry 440 Fall 2008 Lecture 18 O Fructose quot 39 HOCH OHZC O P O HOCH2 O CHZOH f Metabohsm m H OH 5 H Fructose Metabollsm 1n leer OH leer OH OH 7 Fructose D F Wh1ch steps of glycoly51s are bypassed ructose 1 phosphate o H I What is the yield of ATP in the ADP ATP 739 conversion of fructose to pyruvate in OCH J H fOH Glyceraldehyde the liver I CHQOH HCltOH o GHQo h o 39 9 O lI O CH2 Glyceraldehyde 039 00 Dihydroxyacetone 3 phosphate LHZOH phosphate Lecture 18 6 Biochemistry 440 Fall 2008 Lecture 18 Blood glucose Glycogen Gluconeo enesis Animals Pyruvate Lactate Figure 1415 u witneeman and Compan Lecture 18 Disaccharides Glucose 6phosphate A k K Energy Phosphoenol pyruvate CK acl cycle Glucogenic Glycerol amino acids Triacyl glycerols Lehninger Principle af iothernisny Fifth Edition 1003 y Other Glycoproteins monosaccharides Sucrose Plants 3Phospho glycerate co2 xation CoriC Cle Lactate Lactate Lecture 18 Muscle A39I39P produced by glycolysls l39ur rapid uunlnlcliun anmmlt7 Glycogen ATP mm Bland lm lnLv glucose anmis7 gt Glucose ATP Liven ed m synthesis ol39glucuso glnrnnengmwsisi Liming recovery Chicogen Glucose Figure 23 20 Biochemistry 440 Fall 2008 Lecture 18 Glycolysis Glutoneogenesis ATP Glucose Pi Gluconeogenesis is not the reverse of gIYColysis GlyC01y81s VS I Muse mummumwmsphm 7 ADP 6phojsphate H20 39 gt rut ose I Glycoly81s Glucose 2 pyruvate Mg Gingham it Gluconeogenesm 2 pyruvate gt glucose quotWISquot Wfi39sg ggghate quot 39 quot quot Dihydroxyatetone Dihydroxyacetone l Reverse of glycoly81s phospha k phosphae I 2 pyruvate gt ZlGlyceraldehyde 3phosphate glucose ZADP 2Pi 2NAD 2m um I AGIO 2NAD 2NAI 2 NADH 2 H 2 NADH H I 213Bisphosphoglyterate 2 GDP 2 ADP 2 ADP 2 pyruvate 4ATP 2GTP ZNADH 6HZO gt mm Phospho lpymm Epmmkm mm mm glucose 2NAD 4ADP ZGDP 6Pi 2H moxmzzgy 2 3pospogym 0 ZJATP 2mm I 2Pyruvale pyruvatecarhoxylase 2 ATP 22Phospnoglycerate Fig 14716 2 GDP Lecture 18 Biochemistry 440 Fall 2008 2 ADP pyruvate kinase 2 ATP 2 Lactate l I V I I I J HIrIIF Gluconeolgen 318 from pyruvate 2 P P and other 2 AD 23Phos noglycerate Glycer ehyde 3phos ate Glucogenic 22 Phos oglycerate P aminoacids carboxy 2GDBe 2 GDP Phosphoenolpyruvate 2 GTP Oxaloacetate 2 ADP 2 Pyruvate 2 ATP Pyruvate carboxylase Modi ed from Fig 14 16 Lecture 18 Lecture 18 Pyruvate gt oxaloacetate pyruvate Pyruvate carboxylase HCO3 ATP ADP Pi oxaloacetate Biochemistry 440 Fall 2008 Lecture 18 Pyruvate carboxylase mechanism Pyruvate carboxylase mechanism 1 Pyruvate carboxylase b o Hco Pyruvate ATP Site 1 Long biotinylLys quot carboxylase ADP tether moves I 0 gt homotetramer i cozfrom sit a amp L5 gt biotin to site 2 0 o e ix Rf Oc lo 7 o n l Site 2 gtC CCH2 co 0 Pyruvate l o ff 0 gtc c CH2 cf Oxaloacetate Flgure 14 18 Flgure 14 18 0 0 Lecture 18 13 Lecture 18 14 Biochemistry 440 Fall 2008 oxaloacetate Lecture 18 Oxaloacetate gt Phosphoenolpyruvate PEP carboxykinase Ir 0 C OP03239 H CH2 phosphoenolpyruvate 2 ADP pyruvale kinase 2 ATP ll 2 2 GDP PhosphcuenoluyruIateV KEG39hm yk39quotase 2 Pyruvale 2 GT 10xaloacetate 2 ADP pyrume carboxylase 2 ATP PI fructose F t 16bisphosphatase1 rut ose ADP 16 bisphusphate H20 Dihydroxyacetone phosphat Glytolysis Glumneogenesis ATP Glucose Pi VS hexokinase glucose 6phosphataSe Glucose ADP 6phosphate H20 ATP Fructose phosph 6phosphate fructokinasel Dihydroxyacetone phosph Re 2 Glyceraldehyde 3phosphate 2 Pi 2 Pi 2 NAD 2 NAD 2 NADH 2 H 2 NADH H 2 13Bisphosphoglycerate 2 ADP 2 ATP 2 ADP 2 ATP 2 3Phosphoglycerate 2 2Phospnoglycerate Fig 14716 2 GDP Lecture 18 Biochemistry 440 Fall 2008 Lecture 18 g Phosphoenolpyruvate gt glucose the reverse of the same reactions in glycolysis AG39O 163 kImol Glucose 6P H20 gt glucose F1 AG39O 138 kIInol Lecture 18 PEP gt Fructose 16 bisphosphate follows Fructose 16 BP H20 gt Fructose 6 P 13i i Gluconeogenesis is expensive I 2 pyruvate gt 1 glucose using 4ATP 2 GTP using 2 NADH ltgt net yield of glycolysis only 2 ATP and 2 NADH per mol glucose Overall AG 16 kJ mol gt Lecture 18 Biochemistry 440 Fall 2008 Lecture 18 Pentose Phosphate Pathway Produces reducing power I Produces ribose and other pentoses Glucose 6phosphate gt gt C5 gt n39bose SP g Lecture 18 20 Lxm 333 Lxm owx og oobm min 30336 2 283 Nmonmol NI 303E383 Lxm 803mg 825030 L6 L6 3830 A f NmongoLwNI NmOQATwNI NmOQATwNI may owg A 0 o Nmonmolow am 323 n3 32 m6 omouoam Bmaamoagm omo snmm wo Eo omsgummm mmogm OZEEXO oar L M 283 meow in no b28285 Biochemistry 440 Fall 2008 Lecture 18 A simpli ed View of the full 5 Pentose Phosphate Pathway Four mOdQF 9f Operatlon 2mm E 0 E EK Depends on What cells require co2 lt E f I Few Ex f E Mislewg E 9 Lecture 18 24 Biochemistry 440 Fall 2008 Lecture 18 If cells require more ribose If cells require both ribose than NADPH g and NADPH Glucose 671D Glucose 67P Fructose 671D Ribose 571D Ribulose 571D Fructose 167BP Rib 571D Dihydroxyacetone phosphate Glyceraldehyde 371 056 Only the oxidative side of the pentose phosphate pathway Lecture 18 25 Lecture 18 26 Biochemistry 440 Fall 2008 Lecture 18 Cells require mOl e NADPH Cells require NADPH and building than ribose blocks such as pyruvate Glucose 6ephosphate Ribulose SeP Glucose 671D Ribulose 571D Fructose 6ephosphate Ribose SeP Fructose 61D Ribose SeP Fructose 167BP Fructose 1 6ebisphosphate DHAP G371D amino acids DHAP G3P pyruvate acetyl CoA more AT 18 Lecture 18 27 Lecture 18 Biochemistry 440 Fall 2008 Lecture 18 C6 c5 CO 6PHCS CSM NC4N 6 4 c5 cog N Overview of the complete Pentose Phosphate Pathway ZxNADPH C3 CSN cog ZxNADPH 8 C2 C2 Lecture 18 Biochen sh3144OFaH2008 Oxidative Phosphorylation H l The Electron Transport Chain Reading assignment Chapter 191 p 707 722 Lecture 25 Lecture 25 1 it h NADHH Matrix N side The electron transport chain schematic overview Intermembrune space F side E NAD Biochemistry 440 Fall 2008 Lecture 25 NADHtransport into the matrix Malateaspartate shuttle lumrmemhrnne HF Matrix span 39 39NV all NAD r 39 i NAD In Q Malate Malate NADH n NADH oxaloacetate oxaloacetate Glutamate Glutamate Kvmlul m ocketoglutarate 7 aspartate Figure 1929 NADHtransport into the matrix Glycerol 3phosphate shuttle lntermembrane Glycolysis space P side NAD NADH H cytoscllc glycerol 3phosphate dehydrogenase IZHZOH O Glycerol 3 Dihydroxyacetone phosphate phosphate CH2 o CH20H CHOH mitochondrial l glycerol 3phosphate CH2 o dehydrogenase Matrix N side Biochemistry 440 Fall 2008 Lecture 25 Entry of electrons from Boxidation i and Citric acid Cycle g The Ox1dat1on of NADH lntermembrane Glycerol Glycerol l H 02 gt l H20 space P side 339Ph05Phate 3phosphate dehydrogenase c 05 0 10 10 yt I acceptor E donor amp I AGquot 2 nFAE 0 I I AGquot 2 220 kJ mol I Enough to make 7 mole ATP per mole NADH ETFQ oxidoreductase Succinate Fumarate HF I The actual y1eld 18 only 25 mole ATP NADH H AcylCoA dehydrogenase Matrix N side Fatty acyl CoA Lecture 25 6 Lecture 25 EnoylCoA 5 Biochemistry 440 Fall 2008 quot04 Complex I FMN NADH F dehydrogenase 00 Complex II A Succmate Fes E 0 dehydrogenase E T O 4 3 L 08 Electron Transport Chain ETC Lecture 25 Complex 111 Ubiquinone Cytochrome c oxido Cyt C reductase Cyt bcl FeS Complex IV Cyt0chrome oxidase Cyt a a3 Cu Complex 1 NADH dehydrogenase Oxidizes NADH and reduces CoenzymeQ lntermembrane space F side Figure 199 Complex I Lehninger F39n nriple ufBiochemixzm Fifth Edition 2003 w HFreeman and Company Biochemistry 440 Fall 2008 Complex II Succinate dehydrogenase Complex II oxidizes succinate and reduces CoenzyrneQ lntermembrane space P side Phosphatidylethanolamine Matrix N s39de Heme b site Figure 1910 Lecture 25 QH2 cyt c oxidized gt c The complex III mediated Q cycle Oxidation of Oxidation of first Qll2 second QH2 Intermembrane Cytc space F side 2H quot M QH2 Q 2H 1 yr 1 oxidized 39Q 2H cyt 1 reduced OH 2H Q cyt 1 reduced Netequation OH 2cy39c1 oxidized 2Hquot gt Q 2cyt c1 reduced 4H Biochemistry 440 Fall 2008 Complex IV Cytochrome oxidase Complex IV reduces 4 CV 4 oxygen to water on mtermembrane e the matrix side spa P snde 02 Matrix N side 4 H substrate 4H szo Pumped Lecture 25 s Complexes III and IV might associate in membranebound supercomplex llRespirasome a Figure 1 91 5 Lzhningel Principles of niachemiszry Fifth Edition 2008 w H Freeman and Company Lecture 25 Biochemistry 440 Fall 2008 Lecture 25 Summary of electron and proton ow throu h the ETC Intermembrane space P side The free energy released by electron transport is NADH T H conserved as a proton Mamx N sude Figure 1915 gradient and captured Lehninger Principles ol inzhemiitry Fifth Edition zuoswH Freemznnn Company synthase Lecture 25 13 Elucidation of the Electron Transport Chain The order of the electron transport chain Dif cult to study because function of ETC depends on maintaining the structure of the membrane gt Use of inhibitors in intact mitochondria gt Inter ow of electrons from spectra Answer two questions Lecture 25 14 Biochemistry 440 Fall 2008 Lecture 25 UV absorption SPQCtl39a Experimental Plan using Inhibitors of the ETC 10 g 08 Treat mitochondria with antimycin A g 06 Find that NADH Q and cyt b are reduced g 04 Reduced Cyt c1 cyt c cyt a and cyt a3 are oxidized NADH What electron carriers are upstream of the block 0392 the reduced carriers or the oxidized carriers 0 l l l l 220 240 260 280 300 320 340 360 380 Wavelength 11111 b Lecture 25 15 Lecture 25 16 Biochemistry 440 Fall 2008 Lecture 25 139 Electron Transport Chain 704 Complex I FM g Experimental Plan using Inhibitors of the ETC NADH dehydrogenaseFeS Treat mitochondria with amytal I Find that NADH is reduced I Q cyt b cyt c1 cyt c cyt a and cyt a3 are Complex H oxidized cmate Fes Complex 111 E 0 dehydrogenase Ultaiquinon 39 3 Complex V T 04 vmrhmmp 3 oxidase Cyl a 213 Cu Lemma 98 17 Lecture 25 13 Biochemistry 440 Fall 2008 Lecture 25 i Inhibitors of the ETC Complex 1 gt Q gt 111 gt cyt c gt IV Lecture 25 19
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