* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Mechanism of CS, Cont`d
Survey
Document related concepts
Photosynthesis wikipedia , lookup
Light-dependent reactions wikipedia , lookup
Fatty acid metabolism wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Catalytic triad wikipedia , lookup
Butyric acid wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Citric acid cycle wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Biosynthesis wikipedia , lookup
Transcript
King Saud University College of Science Department of Biochemistry Disclaimer • The texts, tables and images contained in this course presentation are not my own, they can be found on: – References supplied – Atlases or – The web Part 3 Coenzyme-Dependent Enzyme Mechanisms Professor A. S. Alhomida 1 2 Thiamin 3 Thiamin Pyrimidine Thiazole Thiamine contains two heterocyclic rings, primidine and thiazole participate in the formation of carbanion-TPP 4 Conversion of Thiamin into Coenzyme Form (TPP) CH3 CH3 N N N H2C NH2 N TPP synthetase H2C H N N ATP S AMP NH2 H S O H3C CH2CH2OH Thiamin H3C CH2CH2O O P O P O O Thiamin pyrophosphate (TPP) 5 O Structure of TPP, Cont’d 6 7 Wet Beri-Beri 8 9 Decarboxylatoion Reactions 10 Decarboxylatoion Reactions • Decarboxlation of carboxylic acid leads to the formation of CO2 and a carbanion • CO2 is a stable molecule, whereas the carbaion is a high-energy molecule that cannot exit for long under the biochemical conditions • The main barrier to decarboxylation is the formation of the carbanion • The decarboxylation will be facilitated when a mechanism exists to stabilize the carbanion produced by decarboxylation 11 Decarboxylation Reaction, Cont’d R1 R2 O C C R3 Carboxylic acid O R2 R1 C C + O R3 O CO2 (stable) Carbanion (unstable) 12 Decarboxylatoion Reactions, Cont’d • How can this be accomplished? • If carbanion is adjacent to an electrondeficient group such as the carbonly group in a ketone, ester, aldehyde or carboxlyic acid • It will be stabilized by delocalization of the electron pair 13 Decarboxylatoion Reactions, Cont’d • b-Keto acids readily undego decarboxylation, whereas the carboxylic acid that have no carbnoly group in the b-position are stable to decarboxylation under physiological conditions • Molecules such as acetic acid, or butyric acid undergo decarboxylation only under extreme conditions such as fusion with solid NaOH 14 Decarboxylatoion Reactions, Cont’d Electron sink O H3 C C H C O O H3 C C H b-Ketoacid O C O H C H3 C C H C + C H H Carbanion O O Enolate ion Carbanion stabilization by delocalization of the electron pair 15 Decarboxylatoion Reactions, Cont’d No Electron sink H3 C H H C C H H O O H3 C C O C O H C + C H O Not b-ketoacid 16 Decarboxylatoion Reactions, Cont’d • How can a decarboxylation reaction be catalyzed? • Decarboxylation of a b-keto acid entails the formation of an enolate ion that is still quite unstable in neutral pH • Any interaction with an enzyme that stabilizes the negative charge will be helpful in the catalyzing decarboxylation 17 Decarboxylatoion Reactions, Cont’d • An enzyme-bound enolate can be stabilized by a positive charged entity such as the proton of an acidic group or the positive charge of metal ion placed near the carbonly oxygen • Stabilization of the enolate lowers the activation energy for the reaction and increases the rate 18 Stabilization of Enolate at Active Site by Acid General acid donates hydrogen bond to the bcarbonly group of a bketo acid General acid donates a H+ to the enolate anion resulting an enol intermediate B B H O H3 C C H H C H b-Keto acid O O O H H3 C C O C C C H O Enol intermediate 19 Stabilization of Enolate at Active Site by Metal Ion Metal ion polarizes hydrogen the b-carbonly group of a b-keto acid via coordination bond Metal ion stabilizes the enolate anion via an electrostatic bond M2+ O H3 C C M2+ H C H b-Keto acid O O O H H3 C C O C C C H O Enolate intermediate 20 Decarboxylatoion Reactions, Cont’d • The enol intermediate is much more stable than the enolate and it is the intermediate in enzymatic reaction rather than the enolate • Conversion of the b-carbonly group into a protonated imine also facilitates the decarboxylation • The pH of an imine is near 7, so that under biochemical conditions the imine-nitrogen can be positively charged and acts as a very effective electron sink 21 Stabilization of Imine Protonated nitrogen of imine R1 H3 C R1 H N C H C H Electron sink H N O H3 C C O b-iminium ion carboxylic acid C Dipolar R1 O N H C H H3 C H C C H H Carbanion imine Enamine 22 + C O Decarboxylatoion Reactions, Cont’d • Decaboxylation of protonated imine, (bcationic imine,b-iminium ion) leads to the formation of an enamine • Enamine is a lower-energy intermediate than an enolate • b-Iminium ion nitrogen carries full positive charge comparing with b-carbonyl group (partially positive charge) • b-Iminium ion facilitates decarboxylation even more effectively than does a b-carbonly group 23 Decarboxylatoion Reactions, Cont’d • b-Iminium ion facilitates decarboxylation even more effectively than does a b-carbonly group • If the keto group of a b-ketoacid is converted into a protonated imine, the rate of decarboxylation will be greatly enhanced • As example of enzymatic decarboxylation via forming imine intermediate: – Acetoacetate decarboxylase 24 Decarboxylatoion Reactions, Cont’d • The enzymes catalyze the dehydrogenations and decarboxylations of b-hydroxy acids do NOT form imines before decarboxylation • They require a divalent cation to facilitate the decarboxylation through coordination with the b-carbonly group via providing positive charge to help stabilize the carbanion intermediate resulting from decarboxylation 25 Decarboxylatoion Reactions, Cont’d • Example of enzymes catalyze b-hydroxy acids: – Malic enzyme – Isocitrate dehydrogenase – 6-phosphogluconate dehydrogenase 26 Decarboxylation of a-Keto Acid 27 Decarboxylation of a-keto Acid • The decarboxylation of a-keto acids occurs frequently in biological systems • It is not obvious that a-keto acids should decarboxlyate readily, because decaroxylation of these acids would NOT produce a stabilized carbanion • These acids undergo a chemical modification before decarboxylation, which converts them into structures resembling b-keto acids 28 Decarboxylation of a-keto Acid, Cont’d • This chemical modification is facilitated by TPP • How does TPP function in decarboxylation of a-keto acids? • TPP can undergo a variety of chemical reactions • It contains a thiazolium ring can easily be deprotonated and forms a Zwitter-ion which reacts as a nucleophile through the carbanion intermediate 29 Comparison Studies R` N 2 H H H S CH3 Thiazolium R` N R CH3 2 O R` N R Oxazolium 2 N H R CH3 Imidazolium 30 Comparison Studies, Cont’d • C-2 oxazolium is more acidic and the oxygen has no d orbitals, however, it is not catalyst • Because C-2 is too stable to add weak electrophilies and unreactive at neutral pH • C-2 imidazolium is very slow to generate carbanion intermediate • Both oxazolium and imidazolium ions are thermodynamic stable at pH 7 31 Comparison Studies, Cont’d • The are NOT suitable for conezyme function as thiazolium ion • The thiazolium ion is the only cone of the three that Is suitable on thermodynamic and kinetic grounds 32 Biochemical Reactions of TPP • TPP is a coenzyme for two types of reactions: • (1) Decarboxylation – (1) Nonoxidative decarboxylation • Yeast pyruvate decarboxylase – (2) Oxidative decarboxylation • a-keto acid dehydrogenases • (2) Transketolaction – Transketolases 33 TPP-Dependent Enzymes O O O TPP, RCHO TPP R COO H a-Keto acid Acetaldehyde TPP, FAD, O2 O O Acetic acid TPP, lipoamide, CoASH, NADH, FAD OH a-Hydroxyacetyl O SCo A Acetyl-CoA 34 Mechanism of Pyruvate Dehydrogenase (PDH) Complex 35 Reaction of PDH Complex, Cont’d 36 Structure of PDH Complex • The transacetylase core (E2) is shown in red, the pyruvate dehydrogenase (E1) in yellow, and the dihydrolipoyl dehydrogenase (E3) in green 37 Structure of Transacelylase • Each red ball represents a trimer of three E2 subunits • Each subunit consists of three domains: (1) lipoamide-binding domain (2) Small domain for interaction with E3 (3) Large transacetylase catalytic domain • All three subunits of the transacetylase are shown in red 38 Structure of PDH Complex • The PDH complex is comprised of multiple copies of three separate enzymes: E1: Pyruvate dehydrogenase (or decarboxylase) (2030 copies) E2: Dihydrolipoyl transacetylase (60 copies) E3: Dihydrolipoyl dehydrogenase (6 copies) 39 Structure of PDH Complex, Cont’d 40 Structure of PDH Complex, Cont’d • The complex also requires 5 different coenzymes: (1) TPP (2) CoA (3) NAD+ (4) FAD+ (5) Lipoamide • TPP, lipoamide and FAD+ are tightly bound to enzymes of the complex whereas the CoA and NAD+ are employed as carriers of the products of PDH complex activity 41 The coenzymes and Prosthetic Groups of PDH Complex Coenzyme Location Function TPP Bound to E1 Decarboxylates Pyr, yielding HE-TPP carbanion Lipoate Covalently linked to Lys on E2 (lipoamide) Accepts HE carbanion from TPP as an acetyl group CoA Coenzyme for E2 Accepts the acetyl group from acetyldihdrolipoamide FAD Bound to E3 Reduced by dihdrolipoamide NAD+ Coenzyme for E3 Reduced by FADH2 42 Structure of PDH Complex, Cont’d • PDH complex is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA • The active sites of all three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme and so on, without diffusion of substrates and products through the solution 43 Lipoic acid • Lipoic acid is a coenzyme found in PDH complex and a-KGDH complex, two multienzymes involved in a-keto acid oxidation • Lipoic acid functions to: – Couple acyl group transfer – Electron transfer during oxidation and decarboxylation of a-ketoacids • No evidence exists of a dietary lipoic acid requirement in humans; therefore it is not considered a vitamin 44 Structure of Lipoamide S • Lipoamide includes a dithiol that undergoes oxidation/ reduction • It acts as a carrier and an redox agent CH2 CH2 S lipoic acid CH O CH2 CH2 CH2 CH2 C NH lysine NH (CH2)4 CH lipoamide C O 2e + 2H+ HS CH2 HS CH CH2 NH O CH2 CH2 CH2 CH2 C NH (CH2)4 CH dihydrolipoamide C 45 O Structure of Lipoamide, Cont’d 1. The carboxyl at the end of lipoic acid's hydrocarbon chain forms an amide bond to the side-chain amino group of a lysine residue of E2 yielding lipoamide S CH2 CH2 S CH lipoic acid O CH2 CH2 CH2 CH2 C NH lysine NH (CH2)4 CH lipoamide C O 2e + 2H+ HS CH2 CH2 HS CH NH O CH2 CH2 CH2 CH2 C NH (CH2)4 CH C 46 O Structure of Lipoamide, Cont’d 2. A long flexible arm, including hydrocarbon chains of lipoate and the lysine R-group, links each lipoamide dithiol group to one of 2 lipoate-binding domains of each E2 S CH2 CH2 S CH lipoic acid O CH2 CH2 CH2 CH2 C NH lysine NH (CH2)4 CH lipoamide C O 2e + 2H+ HS CH2 CH2 HS CH NH O CH2 CH2 CH2 CH2 C NH (CH2)4 CH C 47 O Structure of Lipoamide, Cont’d 3. Lipoate-binding domains are themselves part of a flexible strand of E2 that extends out from the core of the complex S CH2 CH2 S CH lipoic acid O CH2 CH2 CH2 CH2 C NH lysine NH (CH2)4 CH lipoamide C O 2e + 2H+ HS CH2 CH2 HS CH NH O CH2 CH2 CH2 CH2 C NH (CH2)4 CH C 48 O Structure of Lipoamide, Cont’d 4. The long flexible attachment allows lipoamide functional groups to swing between E2 active sites in the core of the complex and active sites of E1 and E3 in the outer shell S CH2 CH2 S CH lipoic acid O CH2 CH2 CH2 CH2 C NH lysine NH (CH2)4 CH lipoamide C O 2e + 2H+ HS CH2 HS CH CH2 NH O CH2 CH2 CH2 CH2 C NH (CH2)4 CH C 49 O Structure of Lipoamide, Cont’d 5. E3 binding protein that binds E3 to E2 also has attached lipoamide that can exchange of reducing equivalents with lipoamide on E2 S CH2 CH2 S CH lipoic acid O CH2 CH2 CH2 CH2 C NH lysine NH (CH2)4 CH lipoamide C O 2e + 2H+ HS CH2 HS CH CH2 NH O CH2 CH2 CH2 CH2 C NH (CH2)4 CH C 50 O Structure of Lipoamide, Cont’d 6. Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase H2O HS R' As O S R' + As HS S R R 7. These highly toxic compounds react with “vicinal” dithiols such as the functional group of lipoamide 51 Formation of TPP-carbanion (Active Form) 52 Formation of TPP-carbanion H N H H B: CH2 H3C N CH3 BH+ O C Glu N N H H S CH2 R H3C B: N N CH3 H O C O O Glu 53 S R Formation of TPP-carbanion, Cont’d Electron sink to stabilize the negative charge H N H CH2 H3C N N CH3 S R O C O Glu 54 Mechanism of PDH Complex 55 Mechanism of PDH Complex CH3 R1 N S R2 Pyruvate decarboxylase TPP carbanion C CH3 C O C O BH O Pyruvate 56 Decarboxylation step CH3 R1 N R1 CH3 N C C OH Enz C O S R2 CH3 CH3 O Tetrahedral intermediate C C OH Enz C O S R2 O Transition state 57 Delocalization of electrons into iminium electron sink R1 CH3 N .. S CO2 R2 CH3 C C CH3 S R2 Electrophile R1 CH3 N OH Enz Nucleophile Dipolar C OH C Enz Carbanion of HETPP Resonance form of hydroxyethyl-TPP 58 S S Enz Electron sink to stabilize the negative charge CH3 Dihydrolipoamide R1 CH3 N C S R2 C OH Enz Hydroxyethyl-TPP S S BH Enz Oxidized (dihydrolipoamide( 59 B: Oxidation and transferring step R1 CH3 CH3 N S H C C O R2 Enz S SH Enz Tetrahedral intermediate CoA SH Dihydrolipoyl transacetylase CH3 N S CH3 CoA-S H R1 R2 C Enz C O B: S BH+ SH TPP Enz Acetyl-dihyrolipoamide (Thioester) 60 Oxidation step HS SH Enz Reduced (dihyrolipoamide) FAD CH3 C NADH + H+ Dihydrolipoyl DH O SCoA Acetyl-CoA FADH2 S NAD+ S Enz Oxidized (dihydrolipoamide( 61 Structure of Dihydrolipoly Transacelyase • Domain structure of the dihydrolipoyl transacetylase (E2) subunit of the PDH complex 62 Structure of Dihydrolipoly Transacelyase, Cont’d • X-Ray structure of a trimer of A. vinelandii dihydrolipoyl transacetylase (E2) catalytic domains 63 Structure of Branched-chain aKeto Acid DH Complex • X-Ray structure of E1 (PDH) from P. putida branched-chain a-keto acid dehydrogenase • The a2b2 heterotetrameric protein • The TPP binds at the interface between a and b subunits 64 Structure of Branched-chain a-Keto Acid DH Complex, Cont’d • X-Ray structure of E1 (PDH) from P. putida branched-chain a-keto acid dehydrogenase • A surface diagram of the active site region • The lipoyl-lysyl armof the E2 lipoyl domain has been model into channel • The TPP-substrate adduct in an enamineTPP form 65 Structure of Dihdrolipoamide DH • X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD+ • The homodimeric enzyme • One subunit is gray and the other is colored according to the domain with its FADbinding domain 66 Structure of Dihdrolipoamide DH, Cont’d • X-Ray structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD and NAD+ • The active site of the enzyme region • The redox-active portions of the bound NAD+ and FAD is shown 67 Mechanism of Dihydrolipoyl DH • Catalytic reaction cycle of dihydrolipoyl dehydrogenase • It is similar to the catalytic reaction cycle of glutathione reductase • However, glutathione reductase uses NADPH instead of NAD+ 68 Catabolism of Branched-Chain Amino Acid O O SCoA O O O NH3 O Isoleucine a-Ketoacid DH Complex O O SCoA O O O NH3 CoA CO2 O Leucine O O NH3 Valine SCoA O O O O 69 Transketolase 70 Reaction of Transketolase CH2OH H CH2OH C O C H C OH transketolase HO H C C H OH CH2-OPO 3H2 D-xylulose-5-phosphate C O HO C H H C OH H C OH CH2-OPO 3H2 H C OH 3-phosphoglyceraldehyde O H C OH H C OH TPP CH2-OPO 3H2 D-ribose-5-phosphate H O C + H CH2-OPO 3H2 septulose-7-phosphate 71 C OH Structure of Transketolase 3- D Structure of yeast 72 Structure of Transketolase • Baker's yeast (Saccharomyces cerevisiae) • The coloring scheme highlights the 2nd structure and reveals that transketolase is a dimer • TPP has been substituted by 2,3'-deazo-thiamin diphosphate which is shown • Ca2+ (blue-gray) can be seen complexed with the diphosphates 73 • Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate 74 Mechanism of Transketolase 75 Mechanism of Transketolase CH3 B: R1 N S R2 R1 CH3 C Enz N H S 1 R2 C CH2OH C O HO C H H C OH BH CH2O P 76 Xylulose-5-phosphate R1 CH3 CH2OH N S R2 H B: C C OH O C H H C OH R1 CH3 N .. S CH2OH C OH C R2 CH2O P Ribose-5-phosphate O H C R1 CH3 H CH2OH C OH N Dihydroxyethyl-TPP S C C OH Glyceraldehyde3-phosphate R2 O H C BH H Ribose-5-phosphate CH2O P C OH 3 CH2O P 77 CH3 R1 CH2OH N C C O HO C H H C OH S R2 H B: 3 CH2O P CH3 R1 N S CH2OH C O O C H H C OH Sedoheptulose-7-phosphate R2 3 CH2O P C Carbanion-TPP 78 Coenzyme A 79 Vitamin B5 (Pantothenic Acid) • Pantothenic acid is also known as vitamin B5 • Pantothenic acid is formed from balanine and pantoic acid • Pantothenate is required for synthesis of CoASH 80 Biosynthesis of CoASH 81 Biosynthesis of CoASH, Cont’d 82 Biosynthesis of CoASH, Cont’d 83 84 85 Function of CoASH • Since CoA is chemically a thiol, it can react with carboxylic acids to form thioesters, thus functioning as an acyl group carrier • It assists in transferring fatty acids from the cytoplasm to mitochondria • A molecule of CoA carrying an acetyl group is also referred to as acetyl-CoA • When it is not attached to an acyl group it is usually referred to as 'CoASH' or 'HSCoA' 86 Acyl Carrier Protein (ACCP) • 4-Phosphopantetheine moiety, linked via its phosphate group to the hydroxyl group of serine, is the active component in another important molecule in lipid metabolism, acyl carrier protein • This is a small protein (8.8 kDa), which is part of the mechanism of fatty acid synthesis • However, the final step in fatty acid synthesis in many types of organism is transfer of the fatty acyl group from ACP to CoA 87 Acyl Carrier Protein Thiol group is the point of attachment to the acyl group being transferred, forming a thioester linkage 88 Structure of CoASH Thiol group is the point of attachment to the acyl group being transferred, forming a thioester linkage Thioester 89 Structure of CoASH, Cont’d 90 Deficiency of Pantothenic Acid • Deficiency of pantothenic acid is extremely rare due to its widespread distribution in whole grain cereals, legumes and meat • Symptoms of pantothenate deficiency are difficult to assess since they are subtle and resemble those of other B vitamin deficiencies 91 Biochemical Features of CoASH Acyl transfer reaction Good leaving group Enolization reaction 92 Activation of Carboxylate Anion by CoASH 93 Activation of Carboxylate Anion, Cont’d Good leaving group O R X Activation C O Carboxylic acid R C Acy transfer Y R O Acceptor Y C O Activated carboxylic group 94 Activation of Carboxylate Anion, Cont’d Good leaving group OH R C BH B: SCoA R OH SCoA + H O B: R C O Tetrahedral intermediate H2O C SCoA O Thioester (Acyl-CoA) 95 Thioesters vs Oxyesters 96 Thioesters vs Oxyesters • Why thioesters in preference to oxyesters? • The enzymatic reaction don’t use oxyesters, but use a thioester derived from CoA • It is advantageous to use thioesters in condensation (Claisen) reactions because the carbonyl carbon atom has more positive character than the carbonly in the corresponding oxyesters 97 Thioesters vs Oxyesters, Cont’d • Thioesters are more readily enolized than oxyesters • Thioesters are more “ketonelike” because of its electronic structures in which the degree of resonce-eletron delocalization from the sulfur atom to the acyl group resulting from overlapping of the occupied p orbitals of sulfur with the acyl p bond is less than that of oxyesters 98 Thioesters vs Oxyesters, Cont’d • The charged-separated resonance form (II) is a smaller contributor to the electronic structure in thioesters than in oxyesters • The reasons for this difference are not fully understood, but one factor may be the larger size of sulfur relative to carbon and oxygen, leading to a poorer energy match for the overlapping orbitals in thioesters relative to oxyesters 99 Thioesters vs Oxyesters, Cont’d • Consider the resonance forms for an oxyester bellow: .. .. O O O O .. .. .. O R O R R C .. O R R C .. R C O R R C + .. 1 I 1 1 1 II III • The contribution from form II tends to decrease the positive charge on the carbon 100 Thioesters vs Oxyesters, Cont’d • However, for thioester, the contribution form II is less important, whereas I and III may be more important than the oxyester • The carbonly carbon of the thioester is more positive than that the oxyester .. .. O R C .. ..S O R1 R C I .. ..S O R1 R O C S R1 .. II R C + .. ..S III 101 R1 Thioesters vs Oxyesters, Cont’d • Positive charge on carbon of the thioester will make it easier for a nucleophilic compound such as carbanion to attack the carbonyl group • It will also make it easier to remove a proton from the adjacent carbon atom to form a carbanion 102 Thioesters vs Oxyesters, Cont’d Easy to be deprotonated H H O C C Not easy to be deprotonated .. S .. R H Thioester H H O C C .. O .. R H More positive charge Oxyester Less positive charge 103 Classification of Mechanism of CoA 104 1. Head Activation Mechanism (Acyl Group Transfer Mechanism) • This reaction involving attack of nucleophilic groups at the acyl carbonyl carbon atom with transfer of the acyl function to the attacking group and release of CoA • This mechanism is called head activation because the end of acyl function nearest to the CoA becomes attached to the nucleophile 105 Head Activation Mechanism (Acyl Group Transfer Mechanism), Cont’d Good leaving group O R C O S CoA R C S Nu + S .. Nu 106 CoA Examples for Head Activation Mechanism • • • • Nu = phosphate: succinly-CoA synthetase Nu = Amine: glucosamine acyl transferase Nu = Water: acetyl-CoA hydrolase Nu = Alcohol: glycerophosphate acetyltransferase • Nu = Thiol: lipoate transferase • Nu = Hydride: acyl-CoA reductase • Nu = Carbanion: b-ketothiolase 107 2. Tail Activation Mechanism (Enolization Mechanism) • This is reaction involving condensation of the alkyl carbon of the acyl-CoA by the alkyl carbon by formation of its carbanion • It is called tail activation because the target group is attached to the acyl function by the end furthest from the CoA 108 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d • This is reaction involving condensation of the alkyl carbon of the acyl-CoA by the alkyl carbon by formation of its carbanion • It is called tail activation because the target group is attached to the acyl function by the end furthest from the CoA 109 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d O O O O C OH C CH3CH O C S O .. CH3CH C S Acyl-CoA a-carbanion CoA 110 CoA 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d • The carbanion on the a-C of the propionlyCoA attacks the bicarbonate to make methylmalonyl-CoA • The facile character of this reaction is attributed to the increased acidity of the thioester compared to the oxyester • Thioester is 100 – 1000 times more acid which means that it has a much greater tendency to undergo proton dissociation at the methylene function immediately adjacent to the sulfur 111 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d • Negative charge that is produced by this dissociation is stabilized by delocalization over the carbonyl group and by the polarizability of the sulfur • Example: Citrate synthetase 112 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism) • Two molecules of acyl-CoA react together • One acyl-CoA undergoes head activation and other undergoes tail activation • The two important steps of the reaction depend on both acyl groups being activated, one for enolization and the other for acylgroup transfer • In the first step, one of the molecules must be enolized by the intervention of a base to remove an a-proton, forming an enolate 113 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d H H B: C O C H S H Acyl-SCoA (Thioester) CoA d+ dB H C O C S CoA H Delocalization of the negative charge 114 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d H O C C S CoA H H O BH + C C CoA S H Carbanion enolate Transition state intermediate 115 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d • The enolate is stabilized by delocalization of its negative charge between the a-carbon and the acyl oxygen atom, making it thermodynamically accessible as an intermediate • The developing charge is also stabilized in the transition state preceding the enolate, so it is also kinetically accessible that means it is readily formed 116 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d • If, by contrast, the acetate anion, it would result in the generation of a second negative charge in the enolate, an energetically and kinetically unfavorable process • Example: b-ketothiolase 117 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d H H B: C O C H Acetate anion H O d+ B O d- H C C O H Unstabilized transition state 118 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d H O C C H Acetate enolate H O BH + C O C O H Kinetically unfavorable intermediate 119 4. Addition Reaction • Reactions involving additions to CoA group • Example: Enoyl-CoA hydratase 120 5. Acyl Group Interchange Reaction • Reactions involving acyl group interchange • Example: Acetoacetyl-CoA transferase 121 Mechanism of Succinyl-CoA Synthetase (Succinyl Thiokinase ) (Head Activation Mechanism) 122 Reaction of Succinyl-CoA Synthetase + DG˚ = - 2.9 kJ/mol 123 Structure of Succinyl-CoA Synthetase • The enzyme is an a2b2 heterodimer; the functional units is one ab pair 124 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) 125 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) Head activation SCoA C O O P OH Pi O His O His (C H2 )2 O COO SCoA OH H O C O (CH2 ) 2 COO N Succinyl-CoA O P N H N N Tetrahedral intermediate It is the displacement of CoA by Pi which generates another high energy compound, succinly-phosphate (phosphoester) 126 BH Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) O O P CoASH O O OH SuccinlylPhosphat C O His C O P (CH2 ) 2 COO His O O N N (CH2 ) 2 OH N BH COO NH B: phosphohistidine His removes the phosphoryl group with the concomitant generation of succinate and phosphohistidine 127 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) His BH O O P O GDP GM O P COO CH2 OH O N N phosphohistidine OH GDP CH2 COO Succinate 128 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) His GTP H N N 129 Mechanism of Citrate Synthtase (Tail Activation Mechanism) 130 Citrate Synthase, Cont’d The monomer of citrate synthase, pictured in the lower frame of the left side of this screen shows the citrate synthase enzyme bound to the two products - citrate 131 Reaction of Citrate Synthase 132 • Two binding sites can be found therein: (1) For citrate or OAA (2) For CoA • The active site contains three key residues: His274, His320, and Asp375 that are highly selective in their interactions with substrates • The enzyme changes from opened to closed with the addition of one of its substrates (such as OAA) 133 The Active Site of Citrate Synthase (including His274, His320, and Asp375 134 CS open State 135 CS Closed State 136 Reaction of Citrate Synthase 137 Reaction of CS, Cont’d OAA E E-OAA Aceyl-CoA E-OAA-Acyl-CoA CoA E-citryl-CoA Citrate E-citrate E Ordered Mechanism 138 CS Stereochemistry 139 Stereochemistry of the CS Reaction 140 Stereochemistry of the CS Reaction, Cont’d 141 Stereochemistry of the CS Reaction, Cont’d 142 Stereochemistry of the CS Reaction, Cont’d 143 Mechanism of Citrate Synthase (Tail Activation Mechanism) 144 Mechanism of CS Deprotonation of a-H+ Asp 375 C C His-320 N H COO C O O O CH2 O SCoA CoA H N COO BH+ O O H N N H C H H C His 274 H H N N C O CH2 H C H N N H C O H H SCoA Enol intermediate COO COO OAA 145 B Mechanism of CS, Cont’d • This conversion begins with the negatively charged oxygen in Asp375 deprotonating acetyl CoA’s a-carbon • This pushes the electron to form a doublebond with the carbonyl carbon, which in turn forces the C=O up to pick up a proton for the oxygen from one of the nitrogens in of His274 to from enol intermediate • It is the rate limiting step of the reaction 146 Mechanism of CS, Cont’d BH+ BH+ C C N O N O H COO O C CH2 COO O H H N N H O H C H H C O H SCoA Enol N H N H COO B O C CH2 COO N H C H N H C O H SCoA Carbanion intermediate 147 BH+ Mechanism of CS, Cont’d • This neutralizes the R-group (by forming a lone pair on the nitrogen) and completes the formation of an enol intermediate • At this point, His274’s amino lone pair formed in the last step attacks the proton that was added to the oxygen in the last step • The oxygen then reforms the carbonyl bond, which frees half of the C=C to initiate a nucleophilic attack to OAA’s carbonyl carbon 148 Mechanism of CS, Cont’d Hydroxlysis of citryl-CoA intermediate B: BH+ H N C O H O SCoA O H C H C SCoA N N H O H C H2 HO H N O COO O H O N H N H2O C C O C H2 HO C COO CH2 CH2 COO COO Citryl-CoA (Thioester) intermediate Tetrahedral intermediate149 N N Mechanism of CS, Cont’d • This frees half of the carbonyl bond to deprotonate one of His320’s amino groups, which neutralizes one of the nitrogens in its R-group • This nucleophilic addition results in the formation of citroyl-CoA intermediate • At this point, a water molecule is brought in and is deprotonated by His320’s amino group and hydrolysis is initiated • One of the oxygen’s lone pairs nucleophilically attacks the carbonyl carbon of citroyl-CoA 150 Mechanism of CS, Cont’d • CS entails the formation of a polarized carbonyl group on OAA and carbanion formation on Acetyl-CoA enhancing production of the condensation product, citrylCoA intermediate • Condensation is followed by the cleavage of the thioester intermediate within the same active site to produce citrate • Each of the important chemical intermediates in the CS reaction is linked to an enzyme conformation change 151 Mechanism of CS, Cont’d BH+ HSCoA C N N O O H N H O H C N O C H2 HO C COO CH2 COO Citrate 152 Mechanism of CS, Cont’d • • • Why is CS suited hydrolyze citryl-CoA but not acetylCoA? How is this discrimination accomplished? CS catalyzes the condensation reaction by bring the substrates into proximity, orienting them, and polarizing certain bonds (1) Acetyl-CoA doesn’t bind to CS until OAA is bound and ready for condensation (2) CS conformation changes and creates binding site for acetyl-CoA (3) The catalytic residues crucial for the hydrolysis of the thioester linkage are not appropriately positioned until citrylCoA is formed and this is happened by induced-fit mechanism to prevent an undesirable side reaction 153