ELEMENTS OF BIOL CHEM
ELEMENTS OF BIOL CHEM BICH 303
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Date Created: 10/21/15
Enzyme Kinetics Dr Leisha Mullins Oct 1 2003 Enzymes Enzymes mediate most of the biochemical reactions occurring in living systems enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction some enzymes are so specific that they catalyze the reaction of only one stereoisomer others catalyze a family of similar reactions Kinetics vs Thermodynamics How Fast gt Reaction rate kinetics The direction gt Reaction Favorability thermodynamics Both are related by Standard Free Energy G The direction a reaction goes depends on its change in standard free energy AG The rate of a reaction depends on its activation energy AG t an enzyme provides an alternative pathway with a lower activation energy Change in Standard Free Energy For a reaction taking place at constant temperature and pressure eg in the body AgB The change in free energy is related to the equilibrium constant Keq for the reaction by AG RT ln Keql Free Energy of Activation Glucose 6O2 6002 6 H20 An enzyme alters the rate of a reaction but not its free energy change or position of equilibrium 0 azmThnmmn wmnnh a Catalyzed Free cnexgy Free energy Pro ts of Leaclion gt Progress of reacL39mIi Activity and Temperature Percent maximum activity Kinetics For the reaction A B the rate of reaction is given by rate equation Rate AA AB AP At At At Rate kA fBg where k is a proportionality constant called the specific rate constant order of reaction the sum of the exponents in the rate equation gtP Cookie Lyase Experiment A cookie Iyase twists an Oreo apart In order to determine the kinetic parameters of the cookie Iyase several experiments are preformed Experiment Oreos per of Oreos desk lysed in 10 sec 1 15 3 2 14 4 3 13 5 4 12 7 5 11 10 6 21 13 7 31 15 8 41 15 9 51 16 Enzyme Catalysis For catalysis to occur the substrate and enzyme must interact substrate 8 a reactant active site the small portion of the enzyme surface where the substrates becomes bound by noncovalent forces eg hydrogen bonding electrostatic attractions van der Waals attractions ES ES enzymesubstrate complex Formation of Enzyme Substrate Complex Two models have been developed to describe formation ofthe enzymesubstrate complex lockandkey model substrate binds to that portion ofthe enzyme with a complementary shape induced fit model binding ofthe substrate induces a change in the conformation of the enzyme that results in a complementary a LockvandL39cy model b imiuccdm model Siihalmlr lawman i yt V n a 39v ACKiV C si e Enzyme complex 2003 Thomson Wadsworth Product Release Product Q 2003 Thomson Wadsworth Cookie Lyase Experiment Saturation Curve Number of cookies lysed in 10 sec Cookie Lyase Experiment 18 16 14 ONBO JOOO 0 1 2 3 4 Number of cookies per desk Enzyme Kinetics At low concentrations ofthe substrate the enzyme is not saturated and the reaction rate is dependent on substrate If substrate concentration is so large that the enzyme is saturated with substrate the reaction rate is NOT dependent on substrate RATHER dependent on enzyme Ci riil dt l39 kinetics of substrate FirstHI tier Linetit39s rate depends on concentration of substrate Initial velocity Vim Substrate concentration iSi gt Michaelis Menten Equation k1 E S k ES 1 k 2gtP rate of product formation k2ES k4 k2 k1 S Vmax S KM S The rate of an enzyme catalyzed reaction v is determined by the constants Km and Vmax and the concentration of the substrate when 8 KM the equation reduces to Important Conclusions of Michaels Menten Kinetics Vm S Vm S vm KM S S S 2 when 8 gtgt KM the equation reduces to vm is vm S 7 V 7 7 7 max KM S S when 8 ltlt KM the equation reduces to lesl Vm s me 5 KM KM KM S Important Conclusions of Michaels Menten Kinetics uumm Unswumtn Reaction velocity V Substrate concentration fSl gt Lineweaver Burk Double Reciprocal Plots it is difficult to determine Vmax experimentally the equation for a hyperbola can be transformed into the equation for a straight line by taking the reciprocal of each side the formula for a straight line is y mx b L KM 1 1 V Vmax S V y m o X b max a plot of 1N versus 1S will give a straight line with slope of KMVmax and y intercept of 1Vmax such a plot is known as a LineweaverBurk double reciprocal plot Lineweaver Burk Double Reciprocal Plots ltH Intercept 1 on x axis K Intercept on yaxis V max 2003 Thomson Wadsworth Cookie Lyase Experiment Experiment Oreos per of Oreos 1 I Oreos 1 I of Oreos desk lysed in 10 per desk lysed in 10 sec sec 1 1 I5 3 5 033 2 1 I4 4 4 025 3 1 I3 5 3 020 4 1 I2 7 2 014 5 1 I1 10 1 010 6 2 I1 13 05 0083 7 3 I1 15 033 0067 8 41 15 025 0067 9 5 I1 16 02 00625 Lineweaver Burk Double Reciprocal Plots 1l Number of cookies lysed in 10 sec Lineweaver Burke of Cookie Lyase Experiment 035 39 I 1l Number of cookies per desk 20 Significance of Km Km is a constant Small Km means tiqht bindinq hiqh Km means weak Mg Useful to compare Km for different substrates for one enzyme Hexokinase Dfructose 15 mM Dglucose 015 mM Useful to compare Km for a common substrate used by several enzymes Hexokinase Dglucose 015 mM Glucokinase Dglucose 20 mM 21 Significance of the Turnover Number kcat The turnover number is a measure of catalytic activity When E is saturated with substrate the observed rate constant is the turnover number turnover number is the number of substrate molecules converted to product per enzyme active site per unit of time Vmax E t kcat Turnover numbr KM Enzyme Function mol Smol E 39ls 391 mmolliter 391 Catalase Conversion of 4 x 107 25 H202 to HzO 02 Carbonic Hydration of C02 1 x 106 12 anhydrase Acetylcholin Regeneration 14 x 10 95 x 102 esterase of acetylcholine Chymotrypsin Proteolytic enzyme 19x 102 66 x 101 Lysozyme Hydrolysis of 05 6 x 103 bacterial cell wall polysaccharides 22 Enzyme Inhibition Reversible inhibitor a substance that binds to an enzyme to inhibit it but can be released competitive inhibitor binds to the active catalytic site and blocks access to it by substrate Irreversible inhibitor a substance that causes inhibition that cannot be reversed usually involves formation or breaking of covalent bonds to or on the enzyme 23 Enzyme Inhibitors quomspm uosmouuaoza Noncome ve inhibitor Fig Newton Oreos and the Cookie Lyase Exp Fig N Oreos of Oreos Exp Fig N Oreos of per per desk lysed in per desk per desk Oreos desk 10sec lysed in 10 sec 1 0 1I5 3 11 1 1I5 15 2 0 1I4 4 12 1 1I4 2 3 0 1I3 5 13 1 1I3 25 4 0 1I2 7 14 1 1I2 4 5 0 1I1 10 15 1 1I1 7 6 0 2I1 13 16 1 2I1 10 7 0 31 15 17 1 3I1 12 8 0 41 15 18 1 41 13 9 0 51 16 19 1 51 14 25 Saturation Curve of a Competitive Inhibitor Fig Newtons are competitive inhibitors of Oreos of Oreos lysed per 10 sec 18 16 14 12 10 DIVhm Oreos and Fig Newtons 0 fig newtons per I 1 newton desk 2 3 4 5 of Oreos per desk 26 Lineweaver Burk Plot of Fig Newtons and Oreos of Oreos lysed per 10 sec Fig Newtons and Oreos O 0 g newtons per I 1 newton desk 25 1 5 05 05 15 25 35 of Oreos per desk 45 55 27 Competitive Inhibitor b 1 4 Z 1 39 39 3 ff K M I r 510 e 1 7 f i i K i p I I KM i 510 e V PC I Vnmx Nu inhibitor present 1 IntelCepl On xax s KM V max 1 KM 1 fl 2003 Thumson Wadsworth Other Types of Inhibition Other Types of Inhibition 30 Lipids and Membranes Dr Leisha Mullins October 152003 Lipids Lipids are substances of biological origin that are soluble in organic solvents Lipids perform three biological functions 1 Lipids in form of a bilayer are essential components of biological membranes 2 Lipids containing hydrocarbon side chains serve as energy stores 3 Many intra and intercellular signaling events involve lipid molecules Lipid Classification Fatty Acids Triacylglycerols Glycerophospholipids Sphingolipids Steroids Fatty Acids The redominate fatty acid rest ues In plants and animals w Wmummml quotquotlquotl i lquot are 016 and 018 W m m in 06 u Polar ma gm Naturally occuring double bonds have the cis configuration Saturated chains paoktightly m and form more rigid organized l39 aggregates ie membranes quot x r a Unsaturated chains bend and pack in a less ordered way with greater potential for motion Nuupnhr m wwwm mmmmm I39nl rhmd gm Fatty Acids TABLE 71 Typical Naturally Occurring Saturated Fatty Acids Number of Melting Point Acid Carbon Atoms Formula C Lauric 1 2 CH3CH2 l GOSH 44 Myristic 14 CH5CH212COZH 58 PalmiLic 16 CH5Hg HCOEH 63 Stearic 18 C113 CH2 MCOQH 71 Arachidic 20 CHZ CH2 mC02H 77 2003 Thomson Wadsworth TABLE 72 Typical Naturally Occurring Unsaturated Fatty Acids Number of Degree of Melting Pom Acid Carbon Atoms U Formula C l almilulcic 16 A 05 39 18 16 Linulcic 13 5 ljnnlenic 18 X 711 Aruchidonic 20 2014 Xquot N H 30 2003 Thomson Wadsworth Triacylglycerols or Triglycerides Fatty acid triesters of glycerol Most contain two or three different types of fatty acid residues animal fats high percent saturated fatty acids residues The melting temperature of a fatty acid varies with degree of saturation and chain length Sciatic rigcicniril Ef cuirznu l l l no on on r o r 1 I 3 T V l 9 Wm 02 20 0 or39 lt 10 lt gt g gt gt gt E gt gt gt n 2 gt 2 8 g M gt gt u K 2 0 lt g x U gt O 8 2 r l 5 lt3 gt lt 3 z gt gt g g lt P r x a gt Myrlsnc Palmllolexc g 2 5mm Trislearin a simple Lriacylglyceml A mixed Lriaq39lglycerol Biological role of Triacylglycerols Triacylglycerols function as energy reservoirs in animals The fat content of normal humans allows them to survive starvation for 2 or 3 months Triacylglycerols function to insulate Olestra carbohydrate based fat substitute Sponification formation of soaps from triacylgerols Snpnni cmion AqucnmNaOH cxymux klcoo x coo 11 Rgcoo RQA OO39 xx Racoo39 wzoo w hmilud Sodium mu ol39 hm arid Polar vs Nonpolar lipids NonPolar Lipids Energy storage 0 Polar lipids are the basis of Bilayers Triacylglycerols Glycerophospholipids Sphingolipids Slorage lipids nonpolar a i Nonpolar ail Polar head An amphipamic lipid Lipid bilayer Glycerophospholipids Glycerophospholipids are the major lipid component of biological membranes oamphiphilic molecules with nonpolar aliphatic tails and polar phosphorylX heads a 0 ll H260CR 0 II HCOC R2 i CHQO P OH O Phosnhatidjc acid 2003 Thomson Wadsworth HC0ltHEhu chCngH 1HIH ZH3 Linolvyl group Phosphatidvl ester JCH270P O X 0 X a hydrogen b clhanolamine c choline d senne c inosilol Structure of X 7H CHrCHrNH3 CHz CHZ MCHQ CHrcx Iif H3 coo mraled fatty acid gv palmidc Unsaturated fany acid eg oleic Name of Glycemphospholipids phosphaudic acid phosphatidylevhanolamine phosphslidylcholine phosphatidylserine phosphatidylinositol Sphingolipids Sphingolipids are major membrane components They are derivatives of the C18 amino alcohol sphingos39ne The double bond in sphingosine is trans Nacyl fatty acid derivatives of sphingosine are known as ceramides Sphingnsine Ceramides Ceramides are the parent compounds of the more abundant sphingolipids 1 Sphingomyelins contain either phosphocholine or phosphoethanolamine The myelin sheath that surrounds and insulates nerve cells is rich in sphingomyelins 0 c 39 NH H H3CH1C7 A c o o H 5 j C P N H HO H 040 5430 Sphingnmyelin N Cerebrosides head group that consists of a single sugar Gangliosides head group with 3 or more sugars one of which is a siaic aci 9 11 Cerebrosides o Fatty acid unit I R CNH Sugar um i H H3CH2C12 CC0 H2 H0 H Cerebroside a glycolipid l I Gangliosides Gangliosides are primarily components of membranes on cell surfaces and constitute 6 of brain lipids Act as receptors for pituitary glycoprotein hormones that regulate physiological functions u o 2003 Thomson Wadsworth 5m GM l N Acely l DGalncose ng39alnclnsaminn nGalacmse DGlllcose CHEOH H o H mm H n 0 n 0 ll H on 1 l u n H OH O H ALAcelyiueuraminidm oialic ac39d Gangliosides whom and G Gangliosidl s Steronds ased on a core structure consisting of three 6membered rings and one 5membered ring all fused together Cholesterol is the most common steroid in animals and precursor for all other steroids in an39ma s Steroid hormones serve many functions in animals including salt balance metabolic function and sexual function m b any can on mmdiul Tuslu lu unu Prugnslurunu ammum rwmswmm Phytosterols Control Serum Cholesterol Levels Te rpenes All lipid molecules biosynthesized from isoprenes Varied biological functions Color and odors associated with plants Vitamin precursors Visual pigments Chloroplast pigments Wsvar myqu away a W 4 mm Eicosanoids Prostaglandins and other eicosanoids are derived from membrane lipids 020 com ounds at low concentration and are involved in the production of pain and fever regulation of blood pressure blood coagulation and reproduction Produced and used locally Arachidonic acid is the most important eicosanoid precursor in humans WW ff W 0 lt A n of o i 7MP WACx H AA WWW H on shammm unfaver EA H o kw Vim D DR 6JA2AAL i w l W 0 an MD Lipid Soluble Vitamins TABLE 73 LipidSoluble Vitamins and Their Functions Vitamin Function Vitamin A Serves as the site I39 the primaiy photochemical reaction in Vision Vitamin D Regulates calcium and phosphonls metabolism Vitamin E Serves as an antioxidant necessary for reproduction in rats and may be necessmy for reproduction in humans Vitamin K Has a regulatory function in blood clotting 2003 Thomson Wadsworth Vitamin A a Cleavage CH3 bCaruleue 0 Enzyme action in liver 2003 Thomson Wadsworth Retinal vitamin A RhodopSI n The best understood role of Vitamin A is its participation in the visual cycle in rod cells the active molecule is retinal vitamin A aldehyde retinal forms an imine with an NH2 group ofthe protein opsin to form the visual pigment called rhodopsin the primary chemical event of vision in rod cells is absorption of light by rhodopsin followed by Isomerization ofthe 11cis double bond to the 11 trans double bond llrisRelinal llhudnpxin Rcauifplmeinm H N r u WNW Rm ofpmxcin nine 5mm hm com o 2003 Thomson Wadsworth 20 Vitamin D A group of structurally related compounds that play a role in the regulation of calcium and phosphorus metabolism the most abundant form in the circulatory system is vitamin D3 Vitamin D 3 Ho 21 Vitamin E Vitamin E is a group of compounds of similar structure the most active is octocopherol an antioxidant traps H00 and R00 radicals formed as a result of oxidation by 02 of unsaturated hydrocarbon chains in membrane phospholipids OH four isoprene units beginning CH3 here and ending at the aromatic ring CH3 CH3 Vitamin E oc Tocopherol 22 Membranes Structures with many cell functlons b wwwmmmm Barrier to toxic molecules lxmri39aqueousrompdruucnl Help accumulate nutrients Carry outenergy transduction Facilitate cell motion Assist in reproduction Modulatesignal transduction lVediate cellcell interactions Hyda39nphilir sinraces Lipid Bilayers The phospholipid bilayer is a fluid matrix The bilayer is a twodimensional solvent Saturated Unsamlalcd Hydruphil k my surfaces head Hydrophobic tails quot X gfgh l c Hydmcarhon Two mil double bomls Membrane Fluidity I39ul M mm group Hydrocarbon an 2003 Thomson Wadsworlh Ordered membrane at Disordered membrane at lower temperature higher temperature Llpld Mobility Lipid do not transfer across a membrane in a process termed transverse diffusion or flip flop However lipids are very mobile in the plane of the membrane and move in a process called lateral diffusion The interior of a bilayer is in constant motion 1 Transverse diffusion flipflop mm mm WW W3 R b Lateral diffusion i 23 mam S lf iww v m MW mm 5 g Bilayer Assymmetry I olar hydr lilnm surfacps QE M We 1 3 e Sphingmnyclin WWJT 9m Ccrebrusida I 0W A De Gunglinside 0 Phosphmcylglycerol Q9 ltnxm1 39 39 39m39A39 wdmpymm l a 3541 2003 Thamsan wadswmh m Membrane Proteins Biological membranes contain proteins and lipids The proteins catalyze reaction mediate the flux of nutrient and waste and participate in relaying external information to the cell Protein to lipid ratios vary with membrane function Lipid rich myelinated membranes surround and insulate nerve axons Protein rich michondrial membrane mediates numerous chemical reaction 28 Types of Membrane Proteins Integral Membrane Proteins Integral or intrinsic proteins are tightly bound to membranes by hydrophobic interactions Integral proteins are amphiphlies Segments immersed in the nonpolar interior Segments that extend into the aqueous environment Lipid Linked Proteins Some proteins are associated with membranes through covalent attachments to lipids that anchor the protein Peripheral membrane proteins Peripheral membrane proteins can be dissociated from the membrane by mild procedures that leave the membrane intact They associate with membranes by binding to the surface 29 2003 Thomson Wadsworth N Mvriswvlation CO 039 S Palmlmvlaxion a munnon Wldlnndh Fluid Mosaic Model Cell exterior Oligosacchan39de protein Phosp holipid Cholesterol Cytosol 2003 Thomson Wadsworth Transport Across Membranes The diffusion of a substance between two sides of a membrane thermodynamically resembles a chemical equilibration Aout lt gt Ain The difference in the a o o 6 9 a 0 o o 0 a a concentration of a 9 Extracellular o 39 q 0 o fluid ECF O quot0 substance on two Sides 0 o 9 go a 9 a 0 of a membrane a 9 a e V 0 generates a chemical potential difference The movement of ions across a membrane also results in a charge difference generating an a do a a 0 J 3 0 0 J a e electrical potential Intracellularfluld CF difference 32 Transport may be mediated or nonmediated Nonmediated transport occurs through simple diffusion where the rate of diffusion depends on the concentration gradient and the substances solubility in the membrane s nonpolar core Water 02 and steroids diffuse readily Mediated transport is classified into two categories depending on the thermodynamics ofthe system 1 Passive mediated transport or facilitated diffusion Molecules flow from high concentrations to low concentrations 2 Active transport A molecule is transported against its concentration gradient This endergonic process must be coupled with an exergonic process to make it thermodynamically favorable Facilitated Diffusion AG negative but proteins assist Solutes only move in the 8 Positive side thermodynamically m favored direction t But proteins may e quotfacilitatequot transport increasing the rates of transport TWO important J 44 JJJ adv 414 1 distinguishing features solute flows only in the favored direction 7 transport displays Mame side saturation kinetics Differences between Simple and Facilitated Diffusion Two important distinguishing features solute ows only in the favored direction transport displays saturation kinetics Facilitated diffusion Passive diffusion GGGGG se Binding Transport WUE gL g gW proteins 3 Egg 3 alternate RRRR H H TTTTTT m behNeen conformations EE E Mediated transport can be W categorized according to the 212 3 stoichiometry of the transport gm 3 process Glucose Transporter Glucose in blood Erymrocyte conc 5 mM Glucose permease a 2m mmmn wmmnn Active Transport Energy input drives transport Some transport must occur such that solutes flow against thermodynamic potential Energy input drives transport Energy source and transport machinery are quotcoupledquot Energy source may be ATP light or a concentration gradient ATP Driven Active Transport active transport is an endergonic process that is generally p coupled to ATP ttiigzi hydrolysis gan 39 blndlng slte 39 Na K ATPase Maintains intracellular Na low and K hi h Crucial for all organs but especially for neural tissue and the brain ATP hydrolysis drives Na out and K in Na inside lt Na outside K inside gt K outside 2mM 145 mM 140 mM 4 M b Lowamnlly Highraflimiy Na39bmding SKES Ktbinuing Slle 2 Q 2 ATP 5 Hignawinnv Lowomniw ADP Na bindm K bmding a sne snes Binding al Aquot coniorrnnuonni pnospnoryinuo39n cha nga omwnro o nspnnuw uansporr nl Nz Binding oi 3 Ni ions 4 4 P M i A A Dissociation Hydrolysis 2 gt 1 Dissocinian 0 Na al Ismnyl conformniaml or K ions 40 binding 0 K phasphaw change Inward transport or K Synaptic neuron AcetylchounE e n re Acuylchoxme hind m recepwr 10n chumcl opens Secondary Active Transport He 39 Ho H Gahncwaidc pcnncaw Fuel e Em wmwn rWIdlwth no Secondary Active Transport lon Gradient Driven Active Transport Systems like the Na Kt ATPase generate electrochemical gradients across membranes that can be used to drive endergonic physiological processes The intestinal epithelium cells take up glucose by a Na dependent symport The uptake of glucose is a secondary active transport because the Na gradient is maintained by the Na K ATPase The Na glucose transport system concentrates glucose inside the cell Na Exterlar Cytosol Glucose 43 Passive Transport of Glucose Glucose is then transported into the capillaries through passive transport Glucose Gutward facing l H 39 Ea glucosebinding Eff glumse site Glumsg Exterior I Plasma 1 2 E mambrana quot 1 39 Cytosol V 1 2 3 Jquot 4 a I Glucose is often given to people suffering from salt and water dehydration since glucose enhances Na resorption which enhances water resorption 44 Glucose Uptake Intestinal lumen Capillaries Naglucose symport GIUCOSE Na KATPase Brush border cell Membrane Receptors Endocylosis 39 W 39 Inside Receptor Overwpply of cholesnerol 2003 Thomson Wadswurth 01016508101 46 Lipid Metabolism Dr Leisha Mullins Dec 2 2003 Lipid Metabolism Fatty Acids are used for energy storage because The carbon in fatty acids mostly CH2 is almost completely reduced so its oxidation yields the most energy possible Fatty acids are not hydrated as mono and polysaccharides are so they can pack more closely in storage tissues Diet and Storage Triglycerides represent the major energy input in the modern American diet Triglycerides are also the major form of stored energy in the body Hormones glucagon epinephrine trigger the release of fatty acids from adipose tissue Dietary LlpldS Dietary lipids are digested by pancreatic lipase Digestion takes place at the lipid water interface Triacylglycerols are water insoluble Digestive enzymes are water soluble Rate of digestion depends on the surface area of the interface Surface area is increased by the peristaltic movements ofthe intestines Bile acids help solubilize the fat globules Pancreatic lipase Triacylglycer l Association wit triacylglycerols OH oH Cholic acid OH OH Bile Salt Micelle Fatty aClds Intestinal Absorption The mixture of mono di and triacylglycerols produced by lipid digestion is absorbed by the intestinal mucosa The chylomicrons are released into the lymphatic system The intestinal lymphatic system drains into the large body veins The blood from the large veins first reach the lungs and the peripheral tissues including the adipose tissue and muscle before reaching the liver LUMEN MUCOSAL CELL 3 Triacylglycerides 393 Other leIds E and proteins H20 j Lipases i To E chylomicrons lymph Fatty acids CE 5Y5tem C Scgt gtTriacylglycerides Monoacylglycerols ggI Hi2 I CZ 512 2 Lipid Transport DIETARY FAT AND CHOLESTEROL EI e sahs 3 Fee Cho es erol Cholese m Intestine Remnam Chylomwcmns chylorm an 0 o 0 90 VLDL Remnants on d CthesterO CapIHanes oapmanes Hydro ysfs of tn acylglycerols in 39cap lanes Cho estero Plasma LCAT Lltpopmiem Key hpase Hydrophiliwayev quotprotein phospthpids etc G ycero Fauy acrds gt Oxwdation m periphevm ussues Triacylg ycerois Cho1eslerol Transport by serum albumm main y in adipose Ussue Lipoprotein Function Lipids are only slightly soluble in aqueous solution so they must be transported in protein complexes Lipoproteins HDL VLDL assemble in the ER of liver cells Chylomicrons form in the intestines LDL not made directly but evolves from VLDL Lipid Metabolism Overview ram mu DECRADAYION mm mu SYNWESi Ii 39 g H u Exx s 5 S mama in pan mm qi may me ismm m mm mm a 2 391 ye i new imamquot R i i Jams i L g x m n Mum mi um MINIla uyi xmup shamed by 10 mm mm Fatty Acid Metabolism Fatty Acids are used for energy storage because The carbon in fatty acids mostly CH2 is almost completely reduced so its oxidation yields the most energy possible Fatty acids are not hydrated as mono and polysaccharides are so they can pack more closely in storage tissues 1 6 H20 D C l cVw cxquotm m 39 f 1 Pa1mitoyLE3adioleoylglyeerol Beta Oxidation fatty acids are degraded in the mitochondria by removal of 20 units the 20 unit released is acetylCoA not free acetate The process begins with oxidation of the carbon that is quotbetaquot to the carboxyl carbon so the process is called quotbetaoxidationquot H2 l 3 fit 53 39 1 Ple quotC39 2 1 n Em C V S H ll rm 3 L I ml 5 Lt Col 5 rCoA S o 0 Beta Oxidation of Fatty Acids Fatty acids must cross into the mitochondria where beta oxidation occurs Fatty Acid Activation Formation of a CoA ester Transport Across the Mitochondrial Membrane Carnitine carries fatty acyl groups across the inner mitochondrial membrane Lipid Degradation betaOxidation of Fatty Acids A Repeated Sequence of 4 Reactions Strategy create a carbonyl group on the betaC J 3 z I v a a It R CHZ CHziCH iLisCDA Acyl SCoAV r A mmng o 1 H Ric zicisCoA T Hs SC A AcylSCnA RCH2CCCSC0A AcerISCoA r Hemyr sum Enuyl SCoA mmlmc 0 AS 0 o unngl gm H H maan R7CH c cufniscm OH 0 2 37Keloacyl SCoA H t Ric gic icHg C SCDA L3 ny1mxyacyr SCoA NADH H L Electron Transferring Flavoprotein Electron inputto electron transport bypasses complex I and II Net result 15 ATP synthesized per electron pairtransferred l l l 0337C zlnif 2rr078ampm H H FAD ETFM ET ubiquinone 1 mum a dehydmgnnm FADH ETFM H o F oxidureduclxaem Mimehondnal slennm W 6 X W Q ETFmbiquinnne mm wdmducmem l ll C a CHn CC C SCnA 2ADF l 2 P H moiho m Steps 2 and 3 Hydration and Oxidation Enoy CoA hydratase Hydroxy acy Co A dehydrogenase Uses NAD o H H R C C COA C C S H2 1 transAzEnoyl CoA H 0 Z Hydration CCCSC A H H LSHydroxyacyl CoA Ho L3 Hydroxyacyl CoA NAD Oxidation H r NADH O 0 H R C C CUA C C 5 H2 5 H H 3Ketoacyl CoA 13 3Keto acyl thiolase Cysteine thiolate on 9 9 enzyme attacks the 3 E w quot39 Rquot a 39 3quot 39 5 99 carbonyl group 1 Carbon Carbon bond f g cleavage yields an E s 0 quot s a go enzyme bound thioester 3 g 3 E3 o 2quot a 2 sum to ii I Thiol group of a new CoA attacks 3 c oquot the shortened chain forming a new 3 gm shorter acylCoA Even though it forms a new thioester the reaction is favorable and drives the three previous 0 reactions 3 R 2 s q B 14 Summary of betaOxidation Repetition of the cycle yields a succession of acetate units Each round of BOxidation yields 1 NADH 1 FADH2 and 1 acetyICoA Each acetyICoA enters TCA generating 1 FADH2 and 3 NADH 1 GTP BOxidation of Stearic acid C18 yields 9 acetyICoAs 8 FADHZ 8 NADH Oxidation of the 9 acetyICoAs yields 9 GTP 27 NADH 9 FADH2 Totals 35 NADH gt 875 ATP 17 FADHZ gt 255 ATP gt 9 9th 8111 7th th 5th 4111 3rd 2nd lst PM l mnurhun nnih 39 39 R x 39 C 8111 7th 6th 5th 4111 3rd 2nd ISL S IDA Cycles 01 Bruxidulinu 1 5 2003 Thomson Wadsworth Synthesis and Utilization of Ketone Bodies I 1rng nt EDA AM u n in ii any cu L Ch mumuymut i mi m mm in V I i Mm m W Hwy 1 X u n a 4 ii i gisinicu Kurt are u M A BHyulmxybul ul H i CH3 c CHzi 200 Hydroxyhunyme NAD NADH unwinmhum ale 0 lehydi ugonmr Ii Cch CH2 c 00 Acetoncemle suwwlcw JKemaryi nA lmnsfel w Succmale ii i CHSC CH2 Q CoA AcelouetyLCoA CoA CoA 39l hiolmu 0 il 3ch CoA 2 Acetyl CDA Fatty Acid Biosynthesis Biosynthesis and Degradation Pathways are Different As in cases of glycolysis gluconeogenesis and glycogen synthesisbreakdown fatty acid synthesis and degradation go by different routes There are four major differences between fatty acid breakdown and biosynthesis Intermediates in synthesis are linked to SH groups of acyl carrier proteins as compared to SH groups of 00A Synthesis in cytosol breakdown in mitochondria Biosynthesis uses NADPHNADP breakdown uses NADHNAD Stereochemistry of the hydration dehydration reaction Biosynthesis 3Dhydroxyacyl breakdown 3Lhydroxyacyl Synthesis vs Degradation Ox dallvs degradauon Synlhesis 0 II E CHz CH2 CH2 CS canier acylCoA FAD mm m N DPH enoyl lt ACP dehydrogenase o H reductase II R CH2 CHCH CS canier enoyl 00A 8 h d H o quotW Wm H 0 Y rWWW ACP hydratase z quot O 2 dehydrase R CIIlz ClH CH2 g S cairia L conngmaum a convlgursuon 8 ketoacyl 3L hydroxyacyl NAD Dohydmamlm Ramaon 30P CoA dehydrogenase 3 El H ACP reducatase R CH2 C CH2 CS carrier co2 Bketoacyl CoA m WW B ketoacyl Thiolase I ACP synthase AcelyvaoA R 39 CH2quot C 8 canier M I 0A 00A or ACP a W c quot39 i degradation synthesis AcelthoA Challenge AcetylCoA in Cytosol What are the sources Amino acid degradation produces cytosolic acetylCoA FA oxidation produces mitochondrial acetylCoA Glycolysis yields cytosolic pyruvate which is converted to acetylCoA in mitochondria Citratemalate pvruvate shuttle tricarboxylate transport system provides cytosolic acetate units AND reducing equivalents for fatty acid synthesis Activation of AcetylCoA Acetate Units are Activated for Transfer in Fatty Acid Synthesis Fatty acids are built from 2 C units acetyICoA Acetate units are activated for transfer by conversion to malonyICoA Decarboxvlation of malonyICoA and reducing power of NADPH drive chain growth Chain grows to 16 carbons Other enzymes add double bonds and more carbons 20 Citratemalatepyruvate shuttle 03 um mooquot NW Mu smart de Map um Wm u mm quotK m quot53 gm mum Nun s co quotanon am A 1 we P HIM u ma 5 3 aha t IB m 7 duh ch as on AM CW an 94quot AcetyICoA Carboxylase The quotACC enzymequot commits acetate to fatty acid synthesis H00 ATP ADP P E bimin Biotinylenzyme Carboxyation of acetyICoA to form malonyl CoA is irreversible committed step ACC uses bicarbonate and ATP AND biotin Ecolienzyme has three subunits Anima enzyme is one polypeptide with all three functions biotin carboxyl carrier biotin carboxylase transcarboxyase Eibiotin 700 Carboxybiotinylenzyme if CHxiCisCoA AcetylCOA if 020 CH2 0780018 E HH MalonylCoA Fatty Acid Synthesis Two fatty acids can be Synthesized simultaneously cu mm r x quotW E 00 It Regulation of FA Synthesis Alosteric modi ers and hormones MalonyICoA blocks the oarnitine acyltransferase and thus inhibits beta oxidation acetyICoA oarboxylase Citrate activates Fatty acyICoAs inhibit Hormones regulate ACC Glucagon activates lipasesinhibits ACC Insulin inhibits lipasesactivates ACC um imszm m KI chumi M m away immein mini mum in veil m may we were ram 11 um mm In mummy unommiim Summay Tnacylglyczml Membrane 11pm lt2 Fwy plds any and synnesls J E J gt Q Chnlasmml lt MerylCM Kazan mm 39 69 5 oxmauan mm phosvnomauon Wuum wawuhnwduyanum w A nwwuunud Electron Transport and Oxidative Phosphorylation Electron Transport Electrons carried by reduced coenzymes are passed through a chain of proteins and coenzymes to drive the generation of a proton gradient across the inner mitochondrial membrane Oxidative Phosphorylation The proton gradient runs downhill to drive the synthesis of ATP e l H B lNTERMLMBRAN e SPACE synthusr ADP ATP H30 Proton Gradient Outer mitochondrial Inner mitochondrial Intermembrane Matrix membrane membrane Electron Lransport leads to proton pumping across the inner mitochondrial mbrane 2003 Thomson Wadsworth Electron Transport Electrons pass from electron donors to electron acceptors NADH Each subsequent electron acceptor wants the electron min more than the previous accep or FAQH2 gt cnb E Standard Reduction Potential A measure of how easily a compound can be reduced The more positive the standard reduction potential the more the compound wants ELECTRONS 5m nt lnulon pumping nupled m At 1 p ndllrrlm 9 95 mmnmm wswunn Standard Reduction Potential NAD2H2e gtNADH H E 0320 12022H2e gtHZO E O816 E for Oxygen is more positive wants to accept electrons E for NAD is more negative wants to donate electron NADH H gtNAD2H2e E 0320 12022H2e gt H20 E 0816 NADH H1202 gtNADHZO A E 1136 Standard Reduction Potential NAD2H2e gtNADH HE O320V ketoglutarate 2H 2 e gt isocitrate E 067 V NAD2H2e gtNADH HE O320V Oxaloacetate 2H 2 e gt Malate E 0166 V TABLE 171 Standard Reduction Potentials for Several Biological Reduction HalfReactions Reduction HalfReaction ED39W i02H39 2FHQO 0816 l c 4 Fe 39 0771 Cyl 15Fe 2 4 Cyl 113Fe39 0350 Cyl MFGquot a39 gt Cyl 1Fe 0290 Cyl AFCquot f A Cyl NFC 0254 Cyl Ft1 F gt Cyt QUE 0220 luQII 39 H F CoQH 0190 COQ 2 H 2 r gt COQH 0060 Cyl MR2 1 quot Cu may 0050 Fumamlc 2 H 2 z 4 SuccinAlc 0051 COQ H39 zquot gt CUQH 39 0030 FAD 2 H 2 e39 gt FADHE 0003 0091 Cyl MFe39 r gt Cyl hiFe 70100 Oxaloacelale 2 H 2 e Malale illl PanvaLe 2 H 2 Aquot Lactate 0185 Acctzlldchyde 2 H 2 e gt Ethanol 70197 2 H 2 Ir FMNHL 0219 FAD2H39 21quotH17ADHE 0219 1 ZBisphosphaglycel39ate 2 H 2 Pquot gt GlyceraldchydeS 290 phosphate 0320 NAD39 2 H 2 a NADH H 0320 NADP 2 H 2 e gt NADPH H 0380 DtrKeloglutarate C03 2 14 2 P gt ISOCiLl leL 0670 Succinate CO 2 H 2 e gt otKetoglularaze HEO 2003 Thumsan Wadsworlh Thermodynamics of Electron Transport AG n F A E n of electrons F Faraday s constant 96 kJ mol391 V1 NADH H1202 gtNADHZO AE 1136 AG 218kJmol The oxidation of NADH by 02 is capable of driving the formation of several moles of ATP Electron Transport Four protein complexes in the inner mitochondrial membrane A lipid soluble coenzyme UQ 000 and a water soluble protein cyt c shuttle between protein complexes Electrons flow through the ETC components in the direction of increasing reduction potentials NADH strong reducing agent E O32 volts O2 terminal oxidizing agent E 082 volts The complexes are laterally mobile within the inner membrane They do not form stable higher order structures and are not present in equimolar concentrations Ymnacwlm nmam Electron Carriers NADH 2 electron transfers Cytochromes and Fe S cluster 1electron transfers A Balm Wm m Methyl g m FMN and COO FMN and COO provide an electron conduit between the 2electron donor NADH and the 1 eectron acceptors the cytochromes a mum momuhmu 1mm mm 1 urqulnnnl rm m a H t N quotYo U ch 1 quot H I 0 mm mama m hy mumonu rm sum 41mm u kmx uimnu arm 0 mm on cm H341 ercHcrcuxtu a lmywnwl um cum 4 my nrl39hiquhmne ukldmdurqul onniuml 11 mm cn Mo n on Cmnw qn an39htwml m um ndiml at new lam LVN on mac an moo u on Cuznzym uc urmlqu ml mama or nymmum arm 11 Complexl NADHCoQ Oxidoreductase Electron transfer from NADH to CoQ Four H transported out per 2 e Complex 1 V 2 mIeeleclmn n ansl39ers MNH F Z 86 e j IL 1 M NADH NAD HQ gt 4H Fcis 2H INTERMEMRRANF SPACE Zonerelecunn v gt mnsrm Complex the overall equation for the reaction of complex I is NADH H EFMN gt NAD EFMNHZ EFMNHZ 2FeS ox gt EFMN 2FeS red 2H FeS red CoQ 2H gt FeS ox CoQH2 NADH H CoQ gt NAD CoQH2 AG 39 81 kJmol 391 this transfer of electrons is strongly exergonic and is sufficient to drive the phosphorylation of ADP ADP Pi gtATP H20 AG 39305kJmol 391 Complex II SuooinateCoQ Reduotase oxidation of FADH2 by CoQ Complex II contains succinate dehydrogenase and 3 other hydrophobic proteins Succinate FAD a Fumarate FADH2 INTERMEM BRANE SPACF Complex II 2 one elsclmn 2 onereleclrou iranslcl s 39 ranslers FeS 69 FAD 3 2 66 Succinate Fumarate 2 He3 2 1GB Complex II Succinatecoenzyme Q oxidoreductase 50 H c0039 2 EFAD gt E EFADH2 on c coo 39OOC H Succinate Fumarate EFADH2 CoQ gt EFAD CoQH2 Succinate CoQ gt Fumarate CoQH2 AG 39135 kJomol 391 the overall reaction is exergonic but not enough to drive ATP production no H is pumped out of the matrix during this step Co Q is a collection point for electrons Inlermembrane I glut m Spa erhnpyhnnz dumdwgcnnse Ew Q NW 3 Succmate oudurrducluw Matrix nul ruA doh drngvnasu Ivauy acyernA Complex III 000 Cytochrome C Oxidoreductase Complex passes electrons from reduced 000 to cytochrome C Transfers electrons to cytochrome c Oxidation of one QH2 is accompanied by the translocation of 4 H across the inner mitochondrial membrane Complex IV Cytochrome C Oxidase 2 Cth Fe2 2 H V2 02 a 2 Cth Fe3 H20 Oxygen is the terminal acceptor of electrons in the electron transport pathway The complete reduction of 02 requires 4 electrons O2 4e 4H gtH20 Complex IV also transports H Complex IV l l l lN39l ERMEMBRANE SPACE onerelectmn lransl urs r 2 onereleclmn 2 Irdmfers CuAHu H I CUB ee 39 l MATRIX l l 2 H Summary of Electron Flow through the ETC lmev membmne Zompiu l Complex m ac 2003 Thomson Wadsworth 2 13quot 02 I lumu39ale Succinam 2003 Thomson Wadsworlh Complex 11 Succinale COQ oxizloreduclase FESm Feslud NADH E FMN Cyum CyLaQX yum 20 NAD39 E FMNiI39 Cy rm Cyuzmd may 0 Complex 1 Complex 111 NADH n CoQHrqmchmmu oxidorcducms Cy II oxidorcducmsc Cytochrome oxidase Oxidative Phosphorylation Electron Transport is Coupled to Oxidative Phosphorylation The free energy released by electron transport is conserved by the creation of an electrochemical H gradient across the inner mitochondrial membrane The electrochemical potential of this gradient is harnessed to synthesize ATP via an ATP synthase Complex V The production of the H gradient is an endergonic process Therefore the dissipation of the gradient is an exothermic process Uncouplers inhibit the phosphorylation of ADP without affecting electron transport examples are 24 dinitrophenol valinomycin and gramicidin A 21 Protonmotive force Outer Inner 2ooaThomson Wadswmm 1 Matrix membmne space membrane c u 9 U u I 39 1 h 39 u u h n v Q q u o 9 in v o 0 1 s Q ATP Synthase or F1FOATPase Proton diffusion through the protein drives A TP synthesis F1 FOATPase is a multisubunit transmembrane protein 450 kD It is composed to two functional units F1 and F0 F0 is a waterinsoluble transmembrane proton channeL F isawatersoluble eri heral membrane 1 I protein 23 Proton Flow thru ATP Synthase Binding Change Mechanism F1h as three interacting catalytic protomers 05B subunits each with a different conformational state L not catalytically active binds ADP and Pi T catalytically active binds ATP 0 a low affinity for substrate Conversion of the L to T synthesis of ATP Conversion of the T to O release of ATP The free energy released with the translocation of a proton is harnessed to interconvert the conformation states The conformational changes are driven by the rotation of the rotor c and y subunits relative to the stator 25 Coupling of Oxidation and Phosphorylation PO ratio the number of moles of Pi consumed in phosphorylation to the number of moles of oxygen atoms consumed in oxidation Phosphorylation P ADP Pi gt ATP H20 Oxidation 0 1202 2H 2e39 gtH20 PO 25 when NADH is oxidized PO 15 when FADH2 is oxidized 27 Uncoupling Oxidative Phosphorylation from Electron Transport Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient Uncouplers are hydrophobic molecules with a dissociable proton They shuttle back and forth across the membrane carrying protons to dissipate the gradient high pH 39 ow pH 0 Oil f O 0 Not r y w 1 lt03 or ll NoZ No if N0 Matrix i Cylosol I N02 2 24Dinitmpllenol DN39P Electron Shuttle Systems Electron Shuttle mechanism transport electrons between mitochondria and cytosol Glycerol phosphate shuttle Malate aspartate shuttle glycolysis in the cytosol found in mammalian produces NADH the transfer of electrons from NADH in the cytosol produces FADH2 in the mitochondria by means of the glycerol phosphate shuttle 15 ATP are produced in the mitochondria for each cystolic NADH kidney liver and heart the transfer of electrons from NADH in the cytosol produces NADH in the mitochondria with the malateaspartate shuttle 25 mitochondrial ATP are produced for each cytosolic NADH 29 Glycerol phosphate shuttle mam Mimdlumlnan mymm phusphan mmmiu my m pumplmm Izllydmgenzw o Dxliydroxwu4ne phmphm a mammsw numm Malateaspartate shuttle Mimclmndrion lunar milncbomlIial membrane Gylosol aKelogluume 00 CH I LAsparmc CH1 I o unglmamm Q mach aparme COO aminmmnsferase LvGIutamalc lecalysis Oxaloacclate ymsouc malale dehydrogenase LMalale Glutamate 60 Manta EH2 00 EH2 HO f H HwN JH 32 coo Oxnloaoemle 00 C S 0 1 COO39 2003 Thumson Wadswoth 31 Glycolysis Dr Leisha Mullins Nov 4 2003 Glycolysis Glucose Catabolism Carried out by all cells GLYCOLYSIS Gl Ten reactions MTP if 2 ADP 739 h same In all cells memm but rates differ 4 Am 2 i Insrzhfgphaw 2NAD Products are 4ATP 2NADH pyruvate 2 waryMe NADH Three possible fates for pyruvate 2 NAD Aerobic Oxidation Anaerobic glycolysis 2mm Anaerobic fermentation h CNADII 30 ADP 2 NAD 2 Lactate 39 2 702 2 Ethanol 2 NAB l 30 ATP to 2003 Thomson Waclsworlh 6 02 6 H20 The Two Phases of Glycolysis First phase converts g 6 C sugar to two 3 C sugar 3 O Second phase produces 2 energy products g mquot E Hwy mm mpxmm Glucose 7 mmumigriwmm ammmwriwwm k Phase 1 ATP 3 Preparative u C phase ATP Fructose 16 bisphosphate 2T Phase 11 nose phosphates ATP generaggg 2mm 2ATP CW 2 ATP rm 2 Pyruvate uan r mmmmmpu 4mm unosuhmylauou I Isomvmzanon WWW y uqun mm mwmwmn m l cleavage 1k u ltgt WW Nwnm f minmm mmmw cm 2003 Thomson Wa swurlh mfg Hexokinase Reaction 1 The first reaction phosphorylation of glucose AG large negative This is a priming reaction ATP is CHon consumed here in order to get more H later HO OH oTraps glucose as glucose6 P which does not diffuse out of the cells But is not the most important site of ATPj m m glueokinssu mr regulation of glycolysis Why ADP cruel 40quot H H HO OH Phosphoglucose Isomerase Reaction 2 OH b Glucose6P to Fructose6P Why does this reaction occur next step phosphorylation at C 1 would be tough for hemiacetal ut easy for primary isomerization activates 03 for cleavage in aldolase reaction Glucose 6phosphal uDglucopyranose form H 0 Tc H z OH HO sC H H C OH H sC OH 6cxizopofa Ghlcosc 6phosphnle openchain form Glam immms szoxi 00 5 oloc112 0 2111011 H0 c H 3 I H H0 if 0H H on H sf OH OH H ECHZDP03 Fructose 6phasphm openchain farm Fructose 6phosphal aDfrucw use fonn Phosphoglucose lsomerase Acid Base Catalysis Starts with a ring opening step Followed by formation of a cisenediolate intermediate and overall proton transfer Ends with ring closure CH20H c H on ll HE C B LO HB H zC OH BH 1 B Gum 2 I HolH Glucos IC OH w HozCH I imam no i H Iwmms H 3 OH gt if 0H H ii OH H 139 on H f OH H OH sH oro ECHZOPDP s 2 3 5CH20P0 Fructose 617hosphniz Glucose 6phosphnte h f openchain form ripen e am am Phosphofructokinase Reaction 3 PFK is the committed step in glycolysis The second priming reaction of glycolysis Committed step and large neg delta G means PFK is highly regulated PFK activity increases when energy status is low PFK activity decreases when energy status is high 6 72 phqsphofmctokmase O POCH 1 V 3 2 O CHZOH p1 Mg 5 H HO 2 ATP gt H I H 4 3 OH 6 HO H 2 OSPOCHZ O CHzopog FructoseGphosphate F6P 5 H Ho 2 ADP H H 4 3 OH HO H Fructosel6bisphosphate BP Aldolase Reaction 4 C6 cleaves to 2 C33 DHAP Glyceraldehyde 3 P Fructose 16bisphosphaw Derived from almylasv Derived fro lucosa carbon glucose carbon 1 H C0 4 II OH 5 CH o s 2 a nihydmxyacemne Glycemldehyde phosphate 3phosphate lmm phosphate munme Aldolase Cont For aldol cleavage to occur between C3 and C4 there must be a carbonyl at C2 and a hydroxyl group at C4 This is the reason that reaction 2 involved the isomerization of G6P to F6P Aldol cleavage of FBP results in two C3 compounds that can be interconverted frame I Ho cen l H IZ Q39D H H COH an KCH20P03 Glycmldehyde 3pllosphale Triosphosphate lsomerase Reaction 5 Only GAP continues along the glycolysis pathway DHAP and GAP are ketose aldose isomers TIM maintains GAP and DHAP at their equilibrium ratio K GAP DHAP 473x 10 2 or DHAP gtgt GAP In glycolysis GAP is being consumed so DHAP is converted to GAP to maintain the equilibrium ratio wwwmu gm Fate of Carbons originating from glucose H yo V izi f zo WISE 17C 9 WC o gt quotW H mc 0H I f zoml CHZOPOP mcmovop 2C 0 Dihydmxyacetone Glyceraldehyde I 3 phosphate H0 3 H 11 0H H c OH 9 3c oc1101gt03 Fructose H71 JOH b39 11 h m 16 up osp a MCHZOPO Glycemldehyde 3phosphate Stage 1 Glycolysis Summary genara on oi high enevgyquot empomd summhval pmspmrylaum rearrangamnl gemnxlon o1 wweneugy39 oanpounu SMDSII BIG IQVBI phaspharyla an m m m um ulu m mm 2003 Thomson Wadswarlh Mun w 339 mun 39 n mwnnx Ilm Indexpump um Adv mh L Wm w MN u Lmuwmm Him I Wu 3m H m um IDO 1 rlxxr co mum I cl I1 Glyceraldehyde 3 Phosphate Dehydrogenase Rxn 6 GAPDH catalyzes the oxidation and phosphorylation of GAP by NAD and Pi Aldehyde oxidation drives the synthesis of the high energy compound 13 bisphosphoglycerate 13 BPG 0 H 0 0H sc sc I oxidation 1 H CI OH mm H20 f HiCiOH NADH H CH20P03239 CH20P03239 0H Acyiphosphaie 0 P 32 C formation C dehydration l H llicH P C OH H20 CH20P03 cmorog GAPH Cont The reaction proceeds Tilifr 39fj e through a thiohemiacetal and acylthioester intermediate 5 o om c i ll IJBisphoshhn gimme IA8P6 Phosphoglycerate Kinase Reaction 7 FEE first ATP is generated in reaction 7 by phosphoglycerate kinase PGK c39atalyzes the transfer of the high ener igggwsphate from 13 BPG yielding ATP and 3phosphogycerate This is referred to as quotsubstratelevel phosphorylationquot Me i ip oi 6Ad nosin2 ii CHgow gquot o 13Bilpholphollyuente Mgquot ADP 7 Mgz o o o no a i i H7227 OH OLE Oi O P O Adenoxine i cnzomg o o 3Pholphollycerau Minn Phosphoglycerate Mutase Reaction 8 Phosphoglycemte C003 I 7 mutase l E H C DH H C OPE13 CHEOP03 CHEOH firPhosphoglyceratc l osphoglyccrate A mutase catalyzes the transfer of a functional group from one position to another on a molecule This is an important step in the preparation to synthesis the next high energy compound Has a unique phosphorylated HIS In Its active Site Enzymrc rfj N Pog PhosphoIIis residue 18 PGM Mechanism The phosphoryl group is transferred to 3PG to form 23 BPG which decomposes to form 2PG and regenerate the phosphorylated enzyme Trace quantities of 23 BPG are required to regenerate phosphoglycerate mutase when it is inactivated m 23Bisphosphoglyoerale inmnncd39mm e ltCHZJA NH3 O e H eel V O P NVNH l 60 H e H c o P e HjOH o 2Phosphoglyceratc Enolase Reaction 9 Enolase catalyzes the dehydration of2PG to PEP 0x 0a 0 0 7 1172127430 OPOQs H1quot H703 AUH 11 H l n 2Phusphoglycerate Phosphoenolpyruvnte ZPG PEP Enolase just rearranges 2PG to a form 39om which more energy can be released in y rolysis The standard free energy for the hydrolysis of 2PG is only 16 kJ mol 1 This is short of the 30 kJ mol 1 necessary to drive the formation of ATP The dehydration o 2PG to PEP by enolase creates a compound PEP capable of the synthesis of AT Pyruvate Kinase The end Pyruvate catalyzes the transfer of the high energyquot phosphate group of PEP to ATP with the formation of pyruvate ADP H Phosphoenolpy39ruvate pyruvalr 0 0 kinasetPKl H J 0 ATP 39Hu Pyruvate The 3 phosphoryl oxygen of ADP nucleophilically attacks the PEP phosphorus atom forming ATP and enolpyruvate Enolpyruvate then tautomerizes to pyruvate This tautomerization is extremely exergonic and represents the majority of the phosphoryl group transfer potential of PEP M 2 039 M 2 f g m l g o o P o 0 o HR 1 l C C o o iDil io Adennsine t K 0 CH2 0 0 PI sphoennl ADP pyruvate PEP Mg2 ATP 1 W I o o 0 o c c 5 L C c 1 7 2 Ktno CH2 0 CH3 Enolpyruvate Pyruvate Glycolysis Stage II Summary 22 Overall Reactions The overall reaction of glycolysis is Glucose 2 NAD 2 ADP 2 Pi gt 2 pyruvate 2 NADH 2 ATP 2 H2O 4 H There is a net gain of 2 ATP per glucose molecule As glucose is oxidized two NAD are reduced to 2 NADH 23 Glycolysis Side Reactions What is the result on the energy yield Arsenate Poisoning GAP AsO4 NAD 7 1 Arseno3PG NADH 0 o 0 1139 0a s c Le H20 Aso cooe H7C0H nmnmm H C OH CH70P03 CH20P03 lAMnoSphosphoglycem 3 Phosphoglyccmte Glyueraldohyde 3phasphate h GAPDH insphuspim 23 BPG J 1 rate 39 o 39 13339 h h 1v Le x p for hemoglobin 15 as 0566 WK i gt H Crown J y IIEOF Uf 3Phosphoglycerate Jusphnsphnr 23Eisphnsphu b glyco u glycerate I POM pimsphabasv 23BPG 2Phosphog yceratc Inherited defects of glycolysis alter the ability of blood to carry oxygen 100 90 He XOMHHSE 30 unimw 70 Normal evanuuyLes Fyruvate kmase ae mem Agggg my mm The Fate of NADH and Pyruvate NADH and Pyruvate are energy two possible fates Aerobic O2 is available NADH is reoxidized in the electron transport pathway making ATP in oxidative phosphorylation Pyruvate enters the citric acid cycle Anaerobic conditions NADH is reoxidized to NAD providing additional NAD for more glycolysis Pyruvate to lactate muscles Pyruvate to ethanol yeast 26 Anaerobic Conditions 2 Alcnhulic fermentation O b mic arjd tnmummian O L HO Hmf39 1 two IHO HPOf cuvo l e 0H H7 0H n on J GSPIquot I can Hgom CHQOPo 7H201 0 i LHLUPUE nGlyceraldehyde 131195 IrGlycemldehyde 13 BPG 3pllosphale 3phosphme NAI NADII l NAn NADH 0 ll mac 100 Km 0H 0 l H 113cm KC 100 umhm lacun dehydmgcme Ammdeh d39 H aenydmgeuue 395 quotV quot Lactate Alcohol Fermentation and Human Consumption of ethanol CO alcohol dehydrogenase z pymvm NADH H NAD decarboxylase CH3 COO CH3 H i CH3ltfHZ 0 1 o 2 0H Pyruvate Acetaldehyde Ethanol CH3CH20H NAD CH3CH0 NADH 11 Ethanol Acetaldchyde A second oxidation step is catalyzed by aldehyde dehydrogenase CH3CHO NADquot cazcoo NADH H Acclaldehyde Acetate