1. 1 Part 5 Coenzyme-Dependent Enzyme Mechanisms Professor A. S. Alhomida 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 King Saud University College of Science Department of Biochemistry

2. 2

3. 3 Thiamin

4. 4 Thiamine contains two heterocyclic rings, primidine and thiazole participate in the formation of carbanion-TPP Pyrimidine Thiazole

5. 5 Conversion of Thiamin into Coenzyme Form (TPP) Thiamin Thiamin pyrophosphate (TPP) TPP synthetase

6. 6 Structure of TPP, Cont’d

7. 7

8. 8 Wet Beri-Beri

9. 9

10. 10 Decarboxylatoion Reactions

11. 11 Decarboxlation of carboxylic acid leads to the formation of CO 2 and a carbanion CO 2 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 Decarboxylatoion Reactions

12. 12 Decarboxylation Reaction, Cont’d Carboxylic acid Carbanion (unstable) CO 2 (stable)

13. 13 Decarboxylatoion Reactions, Cont’d How can this be accomplished? If carbanion is adjacent to an electron- deficient group such as the carbonly group in a ketone, ester, aldehyde or carboxlyic acid It will be stabilized by delocalization of the electron pair

14. 14  -Keto acids readily undego decarboxylation, whereas the carboxylic acid that have no carbnoly group in the  -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 Decarboxylatoion Reactions, Cont’d

15. 15 Decarboxylatoion Reactions, Cont’d  -Ketoacid Carbanion stabilization by delocalization of the electron pair Carbanion Enolate ion Electron sink

16. 16 Decarboxylatoion Reactions, Cont’d Not  -ketoacid No Electron sink

17. 17 How can a decarboxylation reaction be catalyzed? Decarboxylation of a  -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 Decarboxylatoion Reactions, Cont’d

18. 18 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 Decarboxylatoion Reactions, Cont’d

19. 19 Stabilization of Enolate at Active Site by Acid General acid donates hydrogen bond to the  - carbonly group of a  - keto acid General acid donates a H + to the enolate anion resulting an enol intermediate Enol intermediate  -Keto acid

20. 20 Metal ion polarizes hydrogen the  -carbonly group of a  -keto acid via coordination bond Metal ion stabilizes the enolate anion via an electrostatic bond Enolate intermediate  -Keto acid Stabilization of Enolate at Active Site by Metal Ion

21. 21 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  -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 Decarboxylatoion Reactions, Cont’d

22. 22 Stabilization of Imine  -iminium ion carboxylic acid Protonated nitrogen of imine Electron sink Carbanion imine Enamine Dipolar

23. 23 Decaboxylation of protonated imine, (  - cationic imine,  -iminium ion) leads to the formation of an enamine Enamine is a lower-energy intermediate than an enolate  -Iminium ion nitrogen carries full positive charge comparing with  -carbonyl group (partially positive charge)  -Iminium ion facilitates decarboxylation even more effectively than does a  -carbonly group Decarboxylatoion Reactions, Cont’d

24. 24  -Iminium ion facilitates decarboxylation even more effectively than does a  -carbonly group If the keto group of a  -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 Decarboxylatoion Reactions, Cont’d

25. 25 The enzymes catalyze the dehydrogenations and decarboxylations of  -hydroxy acids do NOT form imines before decarboxylation They require a divalent cation to facilitate the decarboxylation through coordination with the  -carbonly group via providing positive charge to help stabilize the carbanion intermediate resulting from decarboxylation Decarboxylatoion Reactions, Cont’d

26. 26 Example of enzymes catalyze b-hydroxy acids: –Malic enzyme –Isocitrate dehydrogenase –6-phosphogluconate dehydrogenase Decarboxylatoion Reactions, Cont’d

27. 27 Decarboxylation of  -Keto Acid

28. 28 Decarboxylation of  -keto Acid The decarboxylation of  -keto acids occurs frequently in biological systems It is not obvious that  -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  -keto acids

29. 29 Decarboxylation of  -keto Acid, Cont’d This chemical modification is facilitated by TPP How does TPP function in decarboxylation of  -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

30. 30 ThiazoliumOxazolium Imidazolium Comparison Studies 222

31. 31 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

32. 32 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 Comparison Studies, Cont’d

33. 33 Biochemical Reactions of TPP TPP is a coenzyme for two types of reactions: (1) Decarboxylation –(1) Nonoxidative decarboxylation Yeast pyruvate decarboxylase –(2) Oxidative decarboxylation  -keto acid dehydrogenases (2) Transketolaction –Transketolases

34. 34 TPP-Dependent Enzymes  -Keto acid Acetaldehyde Acetic acid Acetyl-CoA  -Hydroxyacetyl TPP TPP, RCHO TPP, lipoamide, CoASH, NADH, FAD TPP, FAD, O 2

35. 35 Mechanism of Pyruvate Dehydrogenase (PDH) Complex

36. 36 Reaction of PDH Complex, Cont’d

37. 37 Structure of PDH Complex The transacetylase core (E 2 ) is shown in red, the pyruvate dehydrogenase (E 1 ) in yellow, and the dihydrolipoyl dehydrogenase (E 3 ) in green

38. 38 Structure of Transacelylase Each red ball represents a trimer of three E 2 subunits Each subunit consists of three domains: (1) lipoamide-binding domain (2) Small domain for interaction with E 3 (3) Large transacetylase catalytic domain All three subunits of the transacetylase are shown in red

39. 39 Structure of PDH Complex The PDH complex is comprised of multiple copies of three separate enzymes: E 1 : Pyruvate dehydrogenase (or decarboxylase) (20- 30 copies) E 2 : Dihydrolipoyl transacetylase (60 copies) E 3 : Dihydrolipoyl dehydrogenase (6 copies)

40. 40 Structure of PDH Complex, Cont’d

41. 41 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 Structure of PDH Complex, Cont’d

42. 42 The coenzymes and Prosthetic Groups of PDH Complex CoenzymeLocationFunction TPPBound to E 1 Decarboxylates Pyr, yielding HE-TPP carbanion LipoateCovalently linked to Lys on E 2 (lipoamide) Accepts HE carbanion from TPP as an acetyl group CoACoenzyme for E 2 Accepts the acetyl group from acetyl- dihdrolipoamide FADBound to E 3 Reduced by dihdrolipoamide NAD + Coenzyme for E 3 Reduced by FADH 2

43. 43 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 Structure of PDH Complex, Cont’d

44. 44 Lipoic acid Lipoic acid is a coenzyme found in PDH complex and  -KGDH complex, two multienzymes involved in  -keto acid oxidation Lipoic acid functions to: –Couple acyl group transfer –Electron transfer during oxidation and decarboxylation of  -ketoacids No evidence exists of a dietary lipoic acid requirement in humans; therefore it is not considered a vitamin

45. 45 Structure of Lipoamide Lipoamide includes a dithiol that undergoes oxidation/ reduction It acts as a carrier and an redox agent

46. 46 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 E 2 yielding lipoamide

47. 47 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 E 2

48. 48 Structure of Lipoamide, Cont’d 3.Lipoate-binding domains are themselves part of a flexible strand of E 2 that extends out from the core of the complex

49. 49 4.The long flexible attachment allows lipoamide functional groups to swing between E 2 active sites in the core of the complex and active sites of E 1 and E 3 in the outer shell Structure of Lipoamide, Cont’d

50. 50 5.E 3 binding protein that binds E 3 to E 2 also has attached lipoamide that can exchange of reducing equivalents with lipoamide on E 2 Structure of Lipoamide, Cont’d

51. 51 6.Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase 7.These highly toxic compounds react with “vicinal” dithiols such as the functional group of lipoamide Structure of Lipoamide, Cont’d

52. 52 Formation of TPP-carbanion (Active Form)

53. 53 Formation of TPP-carbanion

54. 54 Formation of TPP-carbanion, Cont’d Electron sink to stabilize the negative charge

55. 55 Mechanism of PDH Complex

56. 56 Mechanism of PDH Complex TPP carbanion Pyruvate Pyruvate decarboxylase

57. 57 Tetrahedral intermediate Decarboxylation step Transition state

58. 58 Resonance form of hydroxyethyl-TPP Carbanion of HETPP Delocalization of electrons into iminium electron sink Dipolar ElectrophileNucleophile

59. 59 Electron sink to stabilize the negative charge Dihydrolipoamide Hydroxyethyl-TPP Oxidized (dihydrolipoamide(

60. 60 Dihydrolipoyl transacetylase TPP Acetyl-dihyrolipoamide (Thioester) Oxidation and transferring step Tetrahedral intermediate

61. 61 Reduced (dihyrolipoamide) Oxidized (dihydrolipoamide( Acetyl-CoA Dihydrolipoyl DH Oxidation step

62. 62 Structure of Dihydrolipoly Transacelyase Domain structure of the dihydrolipoyl transacetylase (E 2 ) subunit of the PDH complex

63. 63 X-Ray structure of a trimer of A. vinelandii dihydrolipoyl transacetylase (E 2 ) catalytic domains Structure of Dihydrolipoly Transacelyase, Cont’d

64. 64 Structure of Branched-chain  - Keto Acid DH Complex X-Ray structure of E 1 (PDH) from P. putida branched-chain  -keto acid dehydrogenase The  2  2 heterotetrameric protein The TPP binds at the interface between  and  subunits

65. 65 X-Ray structure of E 1 (PDH) from P. putida branched-chain  -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 enamine- TPP form Structure of Branched-chain  -Keto Acid DH Complex, Cont’d

66. 66 Structure of Dihdrolipoamide DH X-Ray structure of dihydrolipoamide dehydrogenase (E 3 ) 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 FAD- binding domain

67. 67 X-Ray structure of dihydrolipoamide dehydrogenase (E 3 ) 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 Structure of Dihdrolipoamide DH, Cont’d

68. 68 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 +

69. 69 Catabolism of Branched-Chain Amino Acid Isoleucine Leucine Valine  -Ketoacid DH Complex

70. 70 Transketolase

71. 71 Reaction of Transketolase

72. 72 Structure of Transketolase 3- D Structure of yeast

73. 73 Structure of Transketolase Baker's yeast (Saccharomyces cerevisiae) The coloring scheme highlights the 2 nd structure and reveals that transketolase is a dimer TPP has been substituted by 2,3'-deazo-thiamin diphosphate which is shown Ca 2+ (blue-gray) can be seen complexed with the diphosphates

74. 74 Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate

75. 75 Mechanism of Transketolase

76. 76 Mechanism of Transketolase Xylulose-5-phosphate

77. 77 Glyceraldehyde- 3-phosphate Ribose-5-phosphate Dihydroxyethyl-TPP

78. 78 Carbanion-TPP Sedoheptulose-7-phosphate

79. 79 Coenzyme A

80. 80 Vitamin B 5 (Pantothenic Acid) Pantothenic acid is also known as vitamin B 5 Pantothenic acid is formed from  alanine and pantoic acid Pantothenate is required for synthesis of CoASH

81. 81 Biosynthesis of CoASH

82. 82 Biosynthesis of CoASH, Cont’d

83. 83 Biosynthesis of CoASH, Cont’d

84. 84

85. 85

86. 86 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'

87. 87 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

88. 88 Acyl Carrier Protein Thiol group is the point of attachment to the acyl group being transferred, forming a thioester linkage

89. 89 Structure of CoASH Thiol group is the point of attachment to the acyl group being transferred, forming a thioester linkage Thioester

90. 90 Structure of CoASH, Cont’d

91. 91 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

92. 92 Biochemical Features of CoASH Good leaving group Enolization reaction Acyl transfer reaction

93. 93 Activation of Carboxylate Anion by CoASH

94. 94 Activation of Carboxylate Anion, Cont’d Carboxylic acid Activated carboxylic group Activation Good leaving group Acy transfer Acceptor Y

95. 95 Tetrahedral intermediate Thioester (Acyl-CoA) Good leaving group Activation of Carboxylate Anion, Cont’d

96. 96 Thioesters vs Oxyesters

97. 97 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

98. 98 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  bond is less than that of oxyesters Thioesters vs Oxyesters, Cont’d

99. 99 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 Thioesters vs Oxyesters, Cont’d

100. 100 Thioesters vs Oxyesters, Cont’d Consider the resonance forms for an oxyester bellow: The contribution from form II tends to decrease the positive charge on the carbon

101. 101 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 Thioesters vs Oxyesters, Cont’d

102. 102 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 Thioesters vs Oxyesters, Cont’d

103. 103 Thioesters vs Oxyesters, Cont’d Thioester OxyesterMore positive charge Less positive charge Easy to be deprotonated Not easy to be deprotonated

104. 104 Classification of Mechanism of CoA

105. 105 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 1. Head Activation Mechanism (Acyl Group Transfer Mechanism)

106. 106 Head Activation Mechanism (Acyl Group Transfer Mechanism), Cont’d Good leaving group

107. 107 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:  -ketothiolase Examples for Head Activation Mechanism

108. 108 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

109. 109 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 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d

110. 110 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d Acyl-CoA  -carbanion

111. 111 The carbanion on the  -C of the propionly- CoA 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 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d

112. 112 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 2. Tail Activation Mechanism (Enolization Mechanism), Cont’d

113. 113 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 acyl- group transfer In the first step, one of the molecules must be enolized by the intervention of a base to remove an  -proton, forming an enolate

114. 114 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d ++ -- Delocalization of the negative charge Acyl-SCoA (Thioester)

115. 115 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d Carbanion enolate Transition state intermediate

116. 116 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d The enolate is stabilized by delocalization of its negative charge between the  -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

117. 117 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:  -ketothiolase

118. 118 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d Acetate anion Unstabilized transition state ++ --

119. 119 3. Siamese Twin Reaction (Acyl Transfer and Enolization Mechanism), Cont’d Acetate enolate Kinetically unfavorable intermediate

120. 120 4. Addition Reaction Reactions involving additions to CoA group Example: Enoyl-CoA hydratase

121. 121 5. Acyl Group Interchange Reaction Reactions involving acyl group interchange Example: Acetoacetyl-CoA transferase

122. 122 Mechanism of Succinyl-CoA Synthetase (Succinyl Thiokinase ) (Head Activation Mechanism)

123. 123 Reaction of Succinyl-CoA Synthetase    G ˚ = - 2.9 kJ/mol

124. 124 Structure of Succinyl-CoA Synthetase The enzyme is an  2  2 heterodimer; the functional units is one  pair

125. 125 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism)

126. 126 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) It is the displacement of CoA by P i which generates another high energy compound, succinly-phosphate (phosphoester) PiPi Succinyl-CoA Tetrahedral intermediate Head activation

127. 127 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) His removes the phosphoryl group with the concomitant generation of succinate and phosphohistidine Succinlyl- Phosphat phosphohistidine

128. 128 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism) phosphohistidine Succinate GDP

129. 129 Mechanism of Succinyl-CoA Synthetase (Head Activation Mechanism)

130. 130 Mechanism of Citrate Synthtase (Tail Activation Mechanism)

131. 131 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 Citrate Synthase, Cont’d

132. 132 Reaction of Citrate Synthase

133. 133 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)

134. 134 The Active Site of Citrate Synthase (including His274, His320, and Asp375

135. 135 CS open State

136. 136 CS Closed State

137. 137 Reaction of Citrate Synthase

138. 138 Reaction of CS, Cont’d OAA Aceyl-CoACoACitrate Ordered Mechanism EE-OAA E-OAA-Acyl-CoAE-citrateE-citryl-CoAE

139. 139 CS Stereochemistry

140. 140 Stereochemistry of the CS Reaction

141. 141 Stereochemistry of the CS Reaction, Cont’d

142. 142 Stereochemistry of the CS Reaction, Cont’d

143. 143 Stereochemistry of the CS Reaction, Cont’d

144. 144 Mechanism of Citrate Synthase (Tail Activation Mechanism)

145. 145 Mechanism of CS OAA Deprotonation of  -H + CoA Enol intermediate

146. 146 This conversion begins with the negatively charged oxygen in Asp375 deprotonating acetyl CoA’s  -carbon This pushes the electron to form a double- bond 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 Mechanism of CS, Cont’d

147. 147 Mechanism of CS, Cont’d Enol Carbanion intermediate

148. 148 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 Mechanism of CS, Cont’d

149. 149 Mechanism of CS, Cont’d Citryl-CoA (Thioester) intermediate Hydroxlysis of citryl-CoA intermediate Tetrahedral intermediate

150. 150 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 Mechanism of CS, Cont’d

151. 151 CS entails the formation of a polarized carbonyl group on OAA and carbanion formation on Acetyl-CoA enhancing production of the condensation product, citryl- CoA 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 Mechanism of CS, Cont’d

152. 152 Mechanism of CS, Cont’d Citrate

153. 153 Why is CS suited hydrolyze citryl-CoA but not acetyl- CoA? 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 citryl- CoA is formed and this is happened by induced-fit mechanism to prevent an undesirable side reaction Mechanism of CS, Cont’d