Class Note for BIOC 460 at UA
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EtEIEAEEI V D MtESVEtd Spwtg ZUEIE Redox Reactions in Metabolism Supplemental Reading Key Concepts 7 Reducttnn pptentiats are a measurement pfetectrpn affinity a Cuertzymes prpyide reactiye grpups tnat functipn in enzyme catatysis a Tne pyruyate denydrpgenase cpmptex is a metapptic macnine KEY CONCEPTQUESTIONSIN METABOLIC REDOX REACTIONS wnat dpes tne AE yatue pfa cpupted redpx reactipn tettypu apput etectrpn transferpptentiatf wnat are cpenzymes and npw dp tney functipn in tne pyruyate denydrpgenase reactipnf Btuchemtca Aggtcattmts 0f Cuertz gme Btuchemtstm Thiamin atso catted vitamin 51 is an important enzyme cofactor required for a yariety of metabotic reactions Beriberi is a disease caused by tniamin deficiency resutting in seyere weignt toss and neurotogicat symptoms Peopte tnat eat potisned wnite rice as a sote source of nourisnment can deyetop beriberi because potisned rice tacks tniamin Foods rich in thiamin inctude Watermetoft sunftower seeds btack beans and tniamin enricned grains and breads Reduction potentiats are a measurement of etectron affinit Before we begin discussing tne citrate Cycte tecture 28 we need to describe seyerat biotogicat redox reactions oxtdattonrreductton tnat represent aform of energy conyersion inyotying tne transfer pfetectrpn pairs frpm prganic supstrates tp tne carrier mptecutes NAD and FAD Tne energy ayaitabte from fedox reactions is due to differences in tne etectrpn affinity of two compounds and is an innerent property of eacn motecute based on motecutar structure since etectrons do not ewst free in sotutton etectrons must be passed from one compound to anotner in a coupted fedox reaction Coupted fedox reactions consist of two natf reactions t an oxidation reaction tpss pfetectrpns and 2 a reduction reaction gain pfetectrpns Compounds tnat accept etectrpns are catted oxidants and are reduced in tne reaction wnereas compounds tnat dpnate etectrpns are catted reductants and are said to be UXtdtZE d by toss of etectrons Redox reactions in biocnemistry rarety inyotye motecutar oxygen 02 dtfectty but ratner are cnaracterized by tne toss and gain of electrons from carbon Tne terminotogy of biocnemicat fedox reactions is tne same as tnat used in inorganic cnemistry Namety eacn natf reaction consists of a conjugate redox pair represented by a motecute witn and witnout an etectron 97 For examptet Fe 39Fe3 is a contugate fedox pairin wnicn tneferrous ion Fez39 is tne reductant tnat toses an 97 during oxidation to becorne a ferric ion Fe3 Fez lt7 Fea 97 reductant oxidant Stmttarty tne reductant cuprous ion Cu can be owdized to form tne owdant cupric ion Cuz39 ptus an 97 tn the feacttoft Cu39 ltgt Cu ea reductant oxidant tuftzpages Bloc 4607 Dr Mlesfeld Spring 2008 The two conjugate redox pairs in these reactions are FebFe and Cu lCu We can now combine these two halfreactions into a coupled redox reaction by reversing the direction ofthe Cu reduction reaction such that the 9 functions as the quotcommon intermediatequot shared by the Fe oxidation and Cu reduction halfreactions Fe ltgt Fe e oxidation of Feb Cu e gt Cu reduction of Cu2 Fe Cu lt gt Fe Cu coupled redox reaction The oxidation of Fe and reduction of Cu is a coupled redox reaction we will see in lecture 29 when we examine the Jnction ofthe cytochrome c oxidase complex in the electron transport system mm Number ofer that The combination of glycolysis the cItrate cycle and the Compound Shudme an be donated by electron transport system result in the complete oxidation of glucose to form 002 and H20 by a process called aerobic respiration The e donor is glucose which Jnctions as the reductant and 02 is the e acceptor oxidant that is reduced in the last step ofthe electron transport system to form H20 The two conjugate redox pairs NAD INADH and FADFADHz serve as the ecarriers linking glycolysis to the citrate cycle and electron transport chain It is useful to think Anemldelxyrle of glucose as biochemical quotbatteryquot containing quotl quotquot 39 stored energy in the form of electrons that can be used to synthesize ATP in the mitochondria as a result of n lvllmno proton motive force and oxidative phosphorylation Figure 1 illustrates the relationship between methane twill quot CH4 the most highly reduced form of carbon which has 8 ethat can be donated and carbon dioxide Carbon c a 002 in which all ofthe e shared by the C and O are quot0me tightly associated with the more electronegative O The more electrons a carbon atom has available to donate in a redox reaction the more reduced less oxidized it is Hydrogen is less electronegative than carbon and therefore electrons in CH bonds are considered quotownedquot by the carbon Similarly since oxygen is more electronegative than carbon the electrons in 00 and 00 bonds are all quotovmedquot by the oxygen atom Note that in biological redox reactions often but not always an increase in oxidation state ofa carbon is associated with a decrease in the number of hydrogen atoms Unlike the oxidation of Fe3Wnich simply involves the transfer of one e to Cu redox reactions in the citrate cycle and indeed most all enzymecatalyzed redox reactions involve the transfer of electron pairs 2 e to the electron can39ier molecules NAD and FAD The reduction ofNAD to NADH involves the transfer ofa hydride ion H39 which contains 2 e and 1 H and the release of a proton H into solution NAD 2 e 2 H lt gt NADH H 2 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 In contrast FAD is reduced by sequential addition of one hydrogen 1 e and 1 H at a time to give the fully reduced FADH product FAD1e1H lt gt FADH1eHlt gt FADH Oxidations can also involve a direct combination with oxygen which oxidizes the carbon by pulling e toward the more electronegative O atom Enzymes that catalyze biochemical redox reactions are strictly called oxidoreductases however since most oxidation reactions involve the loss of one or more hydrogen atoms they are often called dehydrogenases We will look at the reduction of the coenzymes NAD and FAD by dehydrogenases in more detail later The two primary energy conversion reactions in metabolism are 1 phosphoryl transfers involving ATP and 2 redox reactions that transfer pairs of electrons between organic compounds and the electron can39iers NAD INADH and FADFADHz As we discussed in lecture 24 the change in standard free energy of a reaction under biochemical conditions AG is a measure of the spontaneity of the reaction in kJmol and re ects the tendency of compound A to be converted to compound B A gt B A negative AG AG lt 0 means the reaction is favored in the direction written from left to right product B will accumulate whereas a positive AG AG gt 0 means the reverse reaction is favorable A will accumulate Figure 2 In redox reactions we use the term reduction potential VOI m e39 E measured in volts V to represent the electron af nity of a given conjugate redox pair Analogous to biochemical standard conditions that define Gibbs Free Energy G 39 25 C pH 7 and 1 M initial concentration of substrates and products the term E refers to the biochemical standard reduction potential under the same conditions Figure 2 illustrates how E values are determined in the laboratory using an apparatus called an electrochemical cell that measures the relative e affinity of a test redox pair compared to that of the hydrogen halfreaction 2H J L 2e lt gt H2 which has been chosen as the standard A For these measurements two halfcells are connected by 1 M Feb M H In equilibrium a type of voltmeter galvanometer and platinum 1M Fe with 1 atm H gas electrodes that measure the movement of electrons from pH 7 one half cell to the other Depending on the relative electron affinity of the test oxidant compared to H the electrons will either move from the hydrogen halfcell toward the test halfcell or from the test halfcell toward the hydrogen halfcell The two halfcells are connected by an agar bridge containing potassium chloride that permits counterion movement to balance the charge The E of oxidants with a higher af nity for electrons than H are recorded as positive E values E gt 0 and oxidants with a lower af nity for electrons than H are have negative E values E lt 0 In the example shown in gure 2 under standard conditions of 25 C and 1 atmosphere of pressure the hydrogen half cell on the right is assigned the arbitrary E value of 000 It can be seen that the voltmeter registers a E value of 0771 volts meaning that the electrons ow from the hydrogen reference cell toward the Fe3Fe2 test cell demonstrating that Fe has a higher affinity for electrons than H 3 of 12 pages Bloc 460 r Dl Mlesfeld Sprll lg 2008 Flgul e 3 M 515mg E0 values that Standard Reduction Potentials of Some Biologically Important have been measured for a HallReactions at 25 c and pH 7 number of conjugate redox pairs in biochemical reactions It can Hallreaction E39 v be seen that 02 isthe most I 08ml potent oxidant in the table with a i 0 i 5039 value of 0816 and will l theremre read39ly accept rulcm 39urrlryallltlc r 035 electrons from all other c 3 n v r I yinnvnmea Fe L e LlLsmaT ltCJlirC m conjugate redox palrs shown 0 2H 2t 739 H O O 7 Whereas OlkEfDQIUfarafe 395 a Cytochromean e a 7 Cylllwmm 7er gt very weak oxidant strongest Cytochrome clre l r 5 34 mm are reductant and will donate CVIUt lIH HH39l39 F e a lytmllrlnlw r IF l 077 eectronsto other conjugate Cylncnmmo MFD a rylocmomo Fc M77 redox pairsinthetable By ULHDUMOHE l 7H 22 mqulllol l H 0025 convention standard reduction I anamlc 7 7l 39 70 a llr Hall 00le potentials are expressed as half L H39 r H aistandald v0l39ldllons pH ul wool reactions Wmenmme direction Crlllull lCnA 7n l 2 alllllwvlCllA coir Daloacolalo 2H 10 e rnalate olou Ufa quotEducquotoquot mama Pwmale PH 7 late Theremrev When EDX39dat39Dn Allillell lll l FH Po gullmmll reaction is written inthe reverse my 2w A 7 FAD direction the sign of the Equot Glulallllulle 7H 2s a 7 reduced glulallllalle 07 Value needsto change 5 4 2H H S accordingly eg from to mer a d 39H gt dln ElmllDWIl39 am The table highlights some ofthe l NAU H e 79 NADH redox reactions we will NADP 39 35 WAD 39 eluaretate 7H 7 a lwdlox lull vale 0146 encounter in metabolism HKetogllllaralo cor 2H l 29 vlsncllraln 038 l l r 29 ll a pll roam The amount of energy available from a coupled redox reaction is directly related to the difference between two reduction potentials and is de ned by the term AE 39 By convention the AE 39 of a coupled redox reaction is determined by subtracting the Equot ofthe oxidant e acceptor from the Equot of the reductant e donor using the following equation AE 39 Ever acceptor Ever donor Moreover the AE 39 for a coupled redox reaction is proportional to the change in free energy AG 39 as described by the equation AG nFAE 39 in which n is the number of electrons transferred in the reaction usually 2 in biochemical redox reactions and Fis the Faraday constant 9648 kJNmol As can be seen by this equation when the difference in reduction potentials for a coupled redox reaction is positive AE 39 gt 0 then the reaction is favorable since AG 39 will be negative Looking back at the de nition of AE 39 this means that for a coupled redox reaction to be favorable the reduction potential of the e acceptor needs to be more positive than that of the e donor 4 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 To see how AG and AE are related we can use the biochemical standard reduction potentials E 39 in gure 3 to calculate the change in biochemical standard free energy AG 39 for the citrate cycle isocitrate dehydrogenase reaction Note that in a spontaneous coupled redox reaction the e ow is from the reductant in the conjugate redox pair with the lower E 39 value more negative toward the oxidant in the conjugate redox pair with the higher E 39 value less negative The two standard half reactions from gure 3 are written below as reduction reactions Note that NAD is the e acceptor less negative and ocketoglutarate is the e donor more negative ocketoglutarate 002 2 e 2 H gt lsocitrate E 39 038 V NAD 2 e 2 H gt NADH H E 39 032 V We can calculate AE 39 for this coupled redox reaction using the equation below AED39 Ewe acceptor 39 Eo39e donor 45 quot E 39NAD EDIlsocitrate AE 39 032 V 038 V 006 v and then convert this AE 39 value to AG 39 using the relationship AG nFAE 39 AG 2 9648 kJmolV 006 v AG 116 kJmol showing that the conversion of isocitrate to ocketoglutarate is a favorable reaction AG 39 lt 0 under standard biochemical conditions Note that since biochemical conditions inside the mitochondrial matrix are not standard in orderto calculate the actual reduction potentials for conjugate redox pairs we need to take into account the concentration of the oxidant e acceptor and reductant e donor using an equation described by Walther Nernst in 1881 E E 39 E o In e acceptor nF e donor In the Nernst equation R is the gas constant 8314 JK mol T is the absolute temperature in Kelvin K n is number of electrons transferred and F is the Faraday constant 9648 kJVmol Coenzymes provide reactive groups that function in enzyme catalysis Pyruvate is a three carbon metabolite derived from glucose or amino acids that must be transported from the cytosol into the mitochondrial matrix before it can serve as a source of reducing power for the cell citrate cycle or as a precursor for glucose synthesis gluconeogenesis Pyruvate that is destined for the citrate cycle or fatty acid synthesis is converted to acetyl CoA by the enzyme pyruvate dehydrogenase As shown in gure 4 acetyl CoA has only two metabolic fates in the cell 1 it can be metabolized by the citrate cycle to convert redox energy to ATP by oxidative phosphorylation or 2 it can be used as a form of stored energy by conversion to fatty acids that are transported to adipocytes fat cells as 5 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 triglycerides Since the pyruvate dehydrogenase reaction is irreversible AG 334 kJmol production of acetylCoA by the pyruvate dehydrogenase reaction is tightly controlled to coordinate energy needs of the cell with the production of acetylCoA Figure 4 This is especially important in animals which lack the necessary Carbohydrates enzymes to convert fats to carbohydrates and therefore cannot reutilized acetylCoA for glucose production when E 7 carbohydrate levels are low Because of this the pyruvate dehydrogenase complex is only fully active in animal cells when G39Ucose carbohydrate sources are plentiful 1 The pyruvate dehyd rogenase complex catalyzes the nale oxidative decarboxylation of pyruvate to form 002 and acetyl Pyruvate dehydrogenase CoA using a ve step reaction mechanism that requires three complex distinct enzymes and ve different coenzymes We begin by first looking at the important role of coenzymes in metabolic ACE4394 reactions specifically NAD FAD CoA thiamine 3 pyrophosphate TPP and lipoic acid which are all utilized in the Citrate Cycle F n A 39dM t b l pyruvate dehydrogenase reaction a y d E 3 mm 2co2 Amino acids can only provide a finite number of chemical Oxidame groups for enzyme reactions and moreover structural Phosphorylation stored energy constraints of the polypeptide backbone restrict the precise In adipocytes positioning orientation of amino acid side groups within the active site However enzyme complexes that bind reactive biomolecules through covalent or noncovalent interactions provide additional reactive groups for catalytic mechanisms These biomolecules are called enzyme cofactors or coenzymes and are often complex organic compounds that are obtained as nutrients in the diet The term vitamin describes organic molecules that are required in the diet but which do not contribute directly to energy conversion through catabolism or do not provide a structural role to other biomolecules Figure 5 lists six coenzymes in metabolism and their role in enzymatic reactions Figure 5 Symptoms of dietary Coenzyme Vitamin Types of reactions Nutrient source deficiency Nicotinarnide adenine Niacin Bg Redox reactions Poultry fish Causes the disease dinuceotide NAD transfer of hydride ion vegetables pellagra Flavin adenine Riboflavin Redox reactions Dairy almonds Causes cheilosis dinuceotide FAD BZ transfer of electrons asparagus swellingcracked lips Coenzyme A CoA Pantothenic Acyl group transfer Chicken yogurt Rarely observed acid B5 avocados Thiamin Thiamin Bi Aldehyde transfer Lentils brown rice Causes beriberi pyrophosphate TPP fortified cereals a LipOiC acid Not a Acyl group transfer Tomatoes broccoli None reported lipoamide vitamin spinach Biocytin biotinlysine Biotin Carboxyl group transfer Breads cooked skin rash hair loss eggs vegetables 6 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 Nicotinamide adenine dinucleotide NAD is derived from the watersoluble vitamin niacin which is also called vitamin Ba NAD and its phosphorylated form NADP are involved in over 200 redox reactions in the cell which are characterized by the transfer of 2 e in the form ofhydride ions H39 Catabolic redox reactions primarily use the conjugate redox pair NAD INADH and anabolic reactions use NADP INADPH The structure of the oxidized and reduced forms of NADP and NADPH respectively are shown in gure 6 Note that the quotquot charge does not refer to the overall charge of the NAD molecule but rather only to the charge on the ring N in the oxidized state Severe niacin de ciency causes the disease pellagra which was rst described in Europe in the early 1700s amongst peasants who relied on cultivated corn as their primary source of nutrition Although it was initially thought that pellagra was caused by an infectious agent in contaminated corn nutritional studies showed that it was due to insuf cient levels of bioavailable niacin in a cornrich diet Interestingly pellagra is rare in Mexico because corn used for tortillas is traditionally soaked in lime solution calcium oxide prior to cooking and this releases niacin from its bound form upon heating Flavin adenine dinucleotide FAD is derived from the watersoluble vitamin ribo avin which is also called vitamin B2 Ribo avin quot is the precursor to FAD and quotH to the related molecule avin mononucleotide FMN both in of which are o en tightly associated with enzymes that catalyze redox reactions W FAD is a coenzyme in the pyruvate dehydrogenase complex and is also covalently bound to a histidine residue in the citrate cycle enzyme succinate dehydrogenase FAD is 7 reduced to FADHQ by the quot transfer of two electrons in un gt11 A r l Figure 6 ii u l 0 tilwu l n ui nzl 17ll l l l l T nu Ull NH l NSF I 1 Ailumnv l N Na lid 0 H l l H mm l mtlmn on on in mm m hm lgmm WNW mm WWW I H n imi39 Hl39ll lhull urn u n H l u nil in w ml Fl mm mum u mm mm the form of hydrogen atoms gure 7 Unlike NAD FAD can accept one electron at a time and form a partially reduced intermediate called a semiquinone FADH39 Foods that have been found to be high in ribo avin include dairy products milk cheese eggs almonds and asparagus Ribo avin like several other vitamins is destroyed by light which is one reason why milk is no longer stored in clear containers 7 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 Coenzyme A CoA is derived from the watersoluble vitamin pantothenic acid which is also called vitamin 85 00A is absolutely essential for life as it is required for energy conversion by the citrate cycle it is Figure 8 also a cofactor in wrvm fatty acid V acetylcholine n i 1 cu i 0 V v M4 heme and M i li39llN i t39H HJ N i S quot 39HJ VHii7fri lL H 5 Cholesterol 0 U llt llu H 0 H Ll biosynthetic uu primary role of 00A quotquot is to function as a carrier molecule for 1 quotquotquotquot quot quotquotquot 39 quotquotquotquotquotquotquot quot acetate units in the form of acetylCoA Figure 8 shows the structure of acetylCoA which consists ofa central pantothenic acid unit that is linked to a functional Smercaptoethylamine group derived from cysteine and to adenosine 35diphosphate The acetate unit is covalently attached to 00A through an activated thioester bond which has a high standard free energy of hydrolysis making it an ideal acyl carrier compound As such attachmentof acetate units to the reduced form of 00A CoASH requires reactions with high AG 39 values for example pyruvate dehydrogenase AG 39 334 kJmol and oc ketoglutarate dehydrogenase AG 39 335 kJmol pathways The quot quot11 I Thiamin pyrophosphate T PP is derived from the QFi ure 9 watersoluble vitamin thiamin or thiamine which is thiiletum also called vitamin B1 The structure of TPP is shown TQM in gure 9 where it can be seen that a carbon atom H I on the thiazole ring isthe functional component of N Z CHZiAICrls 0 the coenzyme involved in aldehyde transfer Thiamin N 4 5 C i foillioi 4 is absorbed in the gut and transported to tissues CH3 H2 LHZ Pi Ii where it is phosphorylated by the enzyme thiamin O kinase in the presence of ATP to form thiamin pyrophosphate TPP and AMP Thiamine pyrophosphate TPP Thiamin de ciency was rst described in Chinese literature over four thousand years ago and is the cause of beriberi a disease characterized by anorexia cardiovascular problems and neurological symptoms Beriberi has been found in populations that rely on white polished rice as a primary source of nutrition milling rice removes the bran which contains thiamin and diets rich in foods that contain the enzyme thiaminase which degrades thiamin during digestion Raw sh contains thiaminase as does African silkworms a favorite food in some Nigerian cultures Cooking these foods destroys the thiaminase and alleviates the symptoms of beriberi aLipoic acid Iipoamide is a coenzyme synthesized in plants and animals as a 68 dithiooctanoic acid The role of ocIipOiC acid in metabolic reactions is to provide a reactive disul de that can participate in redox reactions within the enzyme active site Lipoamide the naturally occurring form of ocIipOiC acid is a covalent linkage of ocIipOiC acid to a lysine samino group on proteins The structure of lipoamide is shown in figure 10 where it can be seen that the long hydrocarbon chain bridging Dlipoic acid and lysine provides a flexible extension to the reactive thiol group 8 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 As illustrated in figure 10 the E2 subunit of the g pyruvate dehydrogenase complex contains the Ii oamide at the end ofa ol e tide tether m P u D W P quot gnaw Wthh functions as a ball and chain that N L I d Amie upon 39 39 In I l I moves the lipoamide back and forth across a y 39es39 en t 50 Aspan in the interior ofthe complex x mi amp C amp C N 2 o rdized Lipoic acid is not considered a vitamin 539 r P C 5 F lipoamide because it is synthesized at measurable levels 0 3 Hydroxyethymp P ruva e In humans however because of Its potential 22 WP dihydmgenase cH Amy to function as an antioxidant in the reduced mp39ex 0 Group form xIipOiC acid is promoted as a nutritional v o n t supplement High levels of OLIIDOIC acnd are In Hf g c c g M g opp quot gggfg lolipoalmde broccoli liver spinach and tomato 0 C L 39 quot I 0 Reduced lipoamide AtchlCM The pyruvate dehydrogenase complex is a metabolic machine The conversion of pyruvate to acetylCoA by the pyruvate dehydrogenase complex is an oxidative decarboxylation reaction that represents another amazing example of protein structure and function The eukaryotic pyruvate dehydrogenase complex contains multiple subunits ofthree different catalytic enzymes that work together as a metabolic machine to carry out the following net reaction Pyruvate CoA NAD gt acetylCoA C02 NADH AG 334 kJmol Coenzymes perform a critical role in the pyruvate dehydrogenase complex by providing a chemical platform for the catalytic reactions Three of the coenzymes are covalently linked to enzyme subunits with TPP attached to the E1 pyruvate dehydrogenase subunit COR lipoamide is the functional component of the Figure 11 E2 dihydrolipoyl E1 transacetylase 0 O I subunit and FAD is covalently bound to the E3 dihydrolipoyl dehydrogenase subunit The two Pyruvate ch C Q FAD r Lipoamide ES l 3 iTPP Ii S HydroxyethyITPP R n Hjc c c TPP NAD NADH FAD SH H S Dihydrolipoamide O I HJC C 3 HS Acetyllipoamide R 3 lHJC CI COA CoA Acetyl CoA 9 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 other coenzymes CoA and NAD are transiently associated with the E2 and E3 complexes respectively The pyruvate dehydrogenase reaction can be broken down into five distinct catalytic steps in which steps 1 2 and 3 lead to the formation of acetylCoA with steps 4 and 5 serving to regenerate the oxidized from of lipoamide and in the process transfer 2 e to NAD figure 11 Step 1 The E1 subunit pyruvate dehydrogenase binds pyruvate and catalyzes a decarboxylation reaction resulting in the formation of hydroxyethylTPP and the subsequent release of 002 Step 2 The hydroxyethylTPP of E1 reacts with the disulfide of the lipoamide group on the N terminal domain of the E2 subunit dihydrolipoyl transacetylase to generate acetyl dihydrolipoamide through a thioester bond Step 3 The E2 lipoamide domain carries the acetyl group from the E1 catalytic site across a 50 A gap in the complex to the E2 catalytic site where it reacts with CoASH to yield acetylCoA and fully reduced dihydrolipoamide Step 4 The lipoamide domain then swings over to the E3 subunit dihydrolipoyl dehydrogenase where it is reoxidized to the disulfide by a transfer of 2 e and 2 H to a disulfide contained on the E3 subunit the dithiol is reoxidized by transferring 2 e and 2 H to the E3linked FAD moiety to transiently form E3FADH2 Step 5 The E3FADH2 coenzyme intermediate is reoxidized in a coupled redox reaction that transfers the 2 e to NAD as a hydride ion H39 leading to the formation of NADH H As shown in figure 12 the E1 E2 and E3 subunits of the mammalian pyruvate dehydrogenase are packed together in a Figure 12 Pyruvate Dehydrogenase Complex huge 400 A diameter sphere with a combined molecular weight of 7800 kDa The colored E1 yellow E2 green and E3 red pyruvate dehydrogenase complex subunits are labeled and the linker region connecting E2 to E1 is shaded gray The stoichiometry of the E1 E23 subunits 22606 is consistent with there being 60 active sites in the pyruvate dehydrogenase complex Figure 13 shows that the lipoamide moiety of the E2 subunit is attached near the end of a 200 amino acid long segment of the protein that functions as both a structural linker connecting the E2 and E1 subunits gray region in figure 16 and as a type of lipoamide quotball and chainquot An important component of the linker region is the E1binding domain that serves as a quotpivotquot for the ball and chain The hydrocarbon extension on the lipoamide Figure 13 Lipoyl domain Elbinding domain E2 Catalytic Domain Lipoamide Amino acid Ball Chain PAVOr Utiker El Lipoamide picks up acetate group from TPP Ball and chain delivers acyl group to CoASH 10 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 moiety itself is sometimes referred to as a quotswinging armquot Because of the relative positioning of E2 and E1 subunits with the pyruvate dehydrogenase complex a single E2 subunit can quotharvestquot hydroxyethyl groups from multiple E1 catalytic sites and deliver them to the E2 subunit The lipoamide group is the workhorse in this catalytic machine and without a fully functional pyruvate dehydrogenase complex the link between glycolysis and the citrate cycle would be broken A naturally occurring inhibitor of lipoamide coenzyme function is the element arsenic As which in the form of arsenite AsOf creates bidentate adducts on dihydrolipoamide as shown in figure 14 Inadvertent ingestion arsenite can lead to an untimely death by irreversibly blocking the catalytic activity of Iipoamidecontaining enzymes such as the pyruvate dehydrogenase and cc ketoglutarate dehydrogenase complexes Figure 14 Figure 15 1mm Ex sawr Oxidized Lipaamide 5Q HydroxyethyerPP 4 H 0H 4 H5 0 lt TPP 4quot 5 5 A W a AFYAHL Pymvatedehydmgenase ornplex is inhibited by Reduced aresenite leading a L pom212 drasric reduttions in aerobit metabolism Chronic arsenic poisoning can come from environmental sources such as arseniccontaminated drinking water or household paints and results in the appearance of ulcerous skin lesions and an increased risk of a variety of cancers While stories abound that arsenic poisoning was used routinely to kill off kings and queens in the Middle Ages and may have even been involved in the death of the exiled French general Napoleon Bonaparte a more tragic example is that of accidental arsenic poisoning of thousands of people in Bangladesh India which has occurred over the last 20 years Since the 1990s it has been documented that millions of people in India have been chronically exposed to toxic levels of arsenic in contaminated drinking water obtained from shallow handpumped wells figure 15 During the 1970s and 1980s UNICEF and other relief organizations helped drill thousands of wells in small Indian villages as an humanitarian effort to circumvent public water supplies that had become biologically contaminated About ten years later when large numbers of villagers in the Ganges delta region developed skin lesions and cancers it was realized that these wells contained water with toxic levels of arsenic Massive efforts were undertaken to close down contaminated wells and to develop purification systems to reduce the arsenic to safe levels in other water supplies Arseniccontaminated drinking water has also been found in Southeast Asia and South America usually near areas that had been extensively mined 11 of 12 pages Bioc 460 Dr Miesfeld Spring 2008 ANSWERS TO KEY CONCEPT QUESTIONS IN THE CITRA TE CYCLE The difference in reduction potential AE for a coupled redox reaction represents the tendency of the reductant in one coniuqate redox pair to donate electrons to the oxidant of the other coniuqate redox pair Since the AE value ofa redox reaction is proportional to the change in free energy AG as described by the equation AG nFAE redox reactions with positive AE values AGltO are more favorable than reactions with negative AE values AGgtO The reduction potential for a given conjugate redox pair is determined experimentally using an apparatus that measures electron flow from one halfcell to another Electrons move through platinum wires from the half cell containing a reductant with low electron af nity toward the halfcell containing an oxidant with high electron af nity In many biochemical redox reactions electrons are transferred in pairs 2 e from one molecule to another sometimes as hydride ions H as is the case with the coenzyme NAD NAD H 2 e ltgt NADH Coenzymes are biomolecules that provide additional functional groups to enzyme active sites and participate in catalytic mechanisms The pyruvate dehydrogenase complex consists of multiple copies of three protein subunits E1 E2 E3 and requires five coenzymes thiamin pyrophosphate TPP lipoamide coenzyme A CoA flavin adenine dinucleotide FAD and nicotinamide adenine dinucleotide NADquot to mediate a fivestep reaction mechanism Most coenzymes are classi ed as vitamins and nutritional de ciencies in some of these important biomolecules can lead to human disease such as beriberi due to thiamin de ciency and pellagra a disease caused by niacin deficiency which leads to decreased levels of NADVNADH 12 of 12 pages
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