1 Part 5 Coenzyme-Dependent Enzyme Mechanisms Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are

1

Part 5 Coenzyme-Dependent Enzyme

MechanismsProfessor A. S. Alhomida

Part 5 Coenzyme-Dependent Enzyme

MechanismsProfessor 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

3

Thiamin

4

Thiamin

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

Pyrimidine Thiazole

5

Conversion of Thiamin into Coenzyme Form (TPP)

NS

NN

CH3

H2C NH2

H3C CH2CH2OH

H

ATPAMP

NS

NN

CH3

H2C NH2

H3C CH2CH2O

H

P

O

O

OP

O

O

O

Thiamin Thiamin pyrophosphate (TPP)

TPP synthetase

6

Structure of TPP, Cont’d

7

8

Wet Beri-Beri

9

10

Decarboxylatoion Reactions

11

• 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

Decarboxylatoion Reactions

12

Decarboxylation Reaction, Cont’d

C C

O

O

R1

R3

R2

CR1

R3

R2

+

O

C

O

Carboxylic acid Carbanion (unstable)

CO2 (stable)

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

• -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


15

C C

O

O

+

O

C

O

O

C

H

H

CH3 C

O

C

H

H

CH3 C

O

C

H

H

CH3


-Ketoacid

Carbanion stabilization by delocalization of the electron pair

Carbanion Enolate ion

Electron sink

16

C C

O

O

+

O

C

O

C

H

H

CH3 C

O

C

H

H

CH3

H

H


Not -ketoacid

No Electron sink

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


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


19

Stabilization of Enolate at Active Site by Acid

C C

O

OO

C

H

H

CH3

B

H

C

O

OO

CH

H

CH3

B

C

H

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

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

C C

O

OO

C

H

H

CH3 C

O

OO

CH

CH3

C

H

M2+ M2+

Stabilization of Enolate at Active Site by Metal Ion

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


22

C C

O

O

+

O

C

O

C

H

H

CH3 CC

H

H

CH3 CC

H

H

CH3

N

R1

HN

HR1 H

N

R1

Stabilization of Imine

-iminium ion carboxylic acid

Protonated nitrogen of imine

Electron sink

Carbanion imine Enamine

Dipolar

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


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


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


26

• Example of enzymes catalyze b-hydroxy acids:– Malic enzyme– Isocitrate dehydrogenase– 6-phosphogluconate dehydrogenase


27

Decarboxylation of -Keto Acid

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

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

NS

RCH3

R` NO

RCH3

R`

HH

N N

RCH3

R`

H

H

Thiazolium Oxazolium Imidazolium

Comparison Studies

2 2 2

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

• 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

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

TPP-Dependent Enzymes

O

COO

O

R

OH

O

H

O

SCoA

O

O

-Keto acidAcetaldehyde

Acetic acid Acetyl-CoA

-Hydroxyacetyl

TPP TPP, RCHO

TPP, lipoamide, CoASH, NADH, FAD

TPP, FAD, O2

35

Mechanism of Pyruvate Dehydrogenase (PDH)

Complex

36

Reaction of PDH Complex, Cont’d

37

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

38

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

39

Structure of PDH Complex

• The PDH complex is comprised of multiple copies of three separate enzymes: E1: Pyruvate dehydrogenase (or decarboxylase) (20-

30 copies)

E2: Dihydrolipoyl transacetylase (60 copies)

E3: Dihydrolipoyl dehydrogenase (6 copies)

40

Structure of PDH Complex, Cont’d

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


42

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 acetyl-dihdrolipoamide

FAD Bound to E3 Reduced by dihdrolipoamide

NAD+ Coenzyme for E3 Reduced by FADH2

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


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

Structure of Lipoamide

S CH2

CH2

CHS

CH2 CH2 CH2 CH2 C NH (CH2)4 CH

NH

C O

O

HS CH2

CH2

CHHS

CH2 CH2 CH2 CH2 C NH (CH2)4 CH

NH

C O

O

2e + 2H+

lipoamide

dihydrolipoamide

lysine lipoic acid • Lipoamide includes a dithiol that undergoes oxidation/ reduction

• It acts as a carrier and an redox agent

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 E2 yielding lipoamide

S CH2

CH2

CHS

CH2CH2CH2CH2CNH(CH2)4 CH

NH

C O

O

HSCH2

CH2

CHHS


NH

C O

O

2e + 2H+

lipoamide

lysine lipoic acid

47


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

CHS


NH

C O

O

HSCH2

CH2

CHHS


NH

C O

O

2e + 2H+

lipoamide

lysine lipoic acid

48


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

CHS


NH

C O

O

HSCH2

CH2

CHHS


NH

C O

O

2e + 2H+

lipoamide

lysine lipoic acid

49

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

CHS


NH

C O

O

HSCH2

CH2

CHHS


NH

C O

O

2e + 2H+

lipoamide

lysine lipoic acid


50

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

CHS


NH

C O

O

HSCH2

CH2

CHHS


NH

C O

O

2e + 2H+

lipoamide

lysine lipoic acid


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

HS

HS

R

S

S

R

R' As O AsR'+

H2O


52

Formation of TPP-carbanion(Active Form)

53

Formation of TPP-carbanion

N

N HH

H3C

CH2 N S

RCH3

H

B:

BH+

N

N H

H3C

CH2 NS

RCH3

H

B:H

Glu

CO

O

Glu

CO

O

54

Formation of TPP-carbanion, Cont’d

N

N HH

H3C

CH2 NS

RCH3

Glu

CO

O

Electron sink to stabilize the negative charge

55

Mechanism of PDH Complex

56

Mechanism of PDH Complex

TPP carbanion

Pyruvate

Pyruvate decarboxylase

C

CH3

R2

R1

N

S

CH3

C O

OC

O

BH

57

Tetrahedral intermediate

Decarboxylation step

C

CH3

R2

R1

N

S

CH3

C O

OC

O

H

Enz

C

CH3

R2

R1

N

S

CH3

C O

OC

O

H

Enz

Transition state

58

Resonance form of hydroxyethyl-TPP

Carbanion of HETPP

Delocalization of electrons into iminium electron sink

Dipolar

ElectrophileNucleophile

C

CH3

R2

R1

N

S

CH3

C OH

Enz

..C

CH3

R2

R1

N

S

CH3

C OH

EnzCO2

59

Electron sink to stabilize the negative charge

C

CH3

R2

R1

N

S

CH3

C OH

S S

Enz

BH

Enz

S S

EnzDihydrolipoamide

Hydroxyethyl-TPP

Oxidized (dihydrolipoamide(

60

C

CH3

R2

R1

N

S

CH3

C O

S S

Enz

H

H

B:

C

CH3

R2

R1

N

S

Enz

EnzCH3

C O

S S

Enz

H

CoA

CoA-S

B:

H

BH+

SH

Dihydrolipoyl transacetylase

TPP

Acetyl-dihyrolipoamide (Thioester)

Oxidation and transferring step


61

FAD

FADH2

NADH

NAD+

+ H+

S S

Enz

HH

CH3

C

SCoA

O

S

Enz

S

Reduced (dihyrolipoamide)

Oxidized (dihydrolipoamide(

Acetyl-CoA

Dihydrolipoyl DH

Oxidation step

62

Structure of Dihydrolipoly Transacelyase

• Domain structure of the dihydrolipoyl transacetylase (E2) subunit of the PDH complex

63

• X-Ray structure of a trimer of A. vinelandii dihydrolipoyl transacetylase (E2) catalytic domains

Structure of Dihydrolipoly Transacelyase, Cont’d

64

Structure of Branched-chain -Keto Acid DH Complex

• X-Ray structure of E1

(PDH) from P. putida branched-chain -keto acid dehydrogenase

• The 22 heterotetrameric protein

• The TPP binds at the interface between and subunits

65

• X-Ray structure of E1

(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

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 FAD-binding domain

67

• 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

Structure of Dihdrolipoamide DH, Cont’d

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

Catabolism of Branched-Chain Amino Acid

Isoleucine

Leucine

Valine

-Ketoacid DH Complex

O

NH3

O

O

NH3

O

O

NH3

O

O

O

O

O

O

O

O

O

O

CoA CO2

SCoA

O

O

SCoA

O

SCoA

70

Transketolase

71

C

CH2OH

C

O

HO H

CH OH

CH2-OPO3H2

C

CH OH

CH OH

CH2-OPO3H2

COH

OHH

D-xylulose-5-phosphate D-ribose-5-phosphate

C

C OH

CH OH

CH2-OPO3H2

H OH

C

H

HHO

C

CH2OH

O

C

C OHH

OH

CH2-OPO3H2

+

septulose-7-phosphate

3-phosphoglyceraldehyde

transketolase

TPP

Reaction of Transketolase

72

Structure of Transketolase

3- D Structure of yeast

73

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

74

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

75

Mechanism of Transketolase

76

Mechanism of Transketolase

C H

CH3

R2

R1

N

S

B:

C

CH3

R2

R1

N

SEnz 1

CH2OH

C OBH

C

OH

OH H

CH

CH2O P

Xylulose-5-phosphate

77

C

CH3

R2

R1

N

S

C

CH3

R2

R1

N

SC OH

BH

C

CH3

R2

R1

N

S..

CH2OH

C

C

OH

O H

CH

CH2O P

OH

HB:

CH2OH

C OH

C

OH

O H

CH

CH2O P

CH2OH

C

OH

O H

CH

CH2O P3

Ribose-5-phosphate

Glyceraldehyde-3-phosphate

Ribose-5-phosphate

Dihydroxyethyl-TPP

78

C

CH3

R2

R1

N

SC O H

B:

CH2OH

C

OH

O H

CH

CH2O P3

H

C O

CH2OH

C

OH

O H

CH

CH2O P3

C

CH3

R2

R1

N

S

Carbanion-TPPSedoheptulose-7-phosphate

79

Coenzyme A

80

Vitamin B5 (Pantothenic Acid)

• Pantothenic acid is also known as vitamin B5

• Pantothenic acid is formed from alanine and pantoic acid

• Pantothenate is required for synthesis of CoASH

81

Biosynthesis of CoASH

82

Biosynthesis of CoASH, Cont’d

83

Biosynthesis of CoASH, Cont’d

84

85

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

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

Acyl Carrier ProteinThiol group is the point of attachment to the acyl group being transferred, forming a thioester linkage

89

Structure of CoASHThiol group is the point of attachment to the acyl group being transferred, forming a thioester linkage

Thioester

90

Structure of CoASH, Cont’d

http://chemistry.gsu.edu/glactone/PDB/Cofactor/coa.pdb

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

Biochemical Features of CoASH

Good leaving group

Enolization reactionAcyl transfer reaction

93

Activation of Carboxylate Anion by CoASH

94

Activation of Carboxylate Anion, Cont’d

CR

O

O

CR

X

O

CR

Y

O

Carboxylic acid Activated carboxylic group

Activation

Good leaving group

Acy transfer

Acceptor Y

95


Thioester (Acyl-CoA)

Good leaving group

Activation of Carboxylate Anion, Cont’d

CR

O

O

+ SCoA

H

B:

H

CR SCoA

BHH

O

O

CR SCoA

OH2O

B:

96

Thioesters vs Oxyesters

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

• 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

• 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


100


• Consider the resonance forms for an oxyester bellow:

• The contribution from form II tends to decrease the positive charge on the carbon

CR

O

R1 CR

O

R1 ..

..

CR

O

R1

....

CR

O

R1

..+..

I II III

..

.. OO O O

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


CR

O

R1 CR

O

R1 ..

..

CR

O

R1

....

CR

O

R1

..+..

I II III

S S..

S S....

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


103


C

O

R....SC

H

H

H C

O

R....OC

H

H

H

Thioester OxyesterMore positive charge

Less positive charge

Easy to be deprotonated

Not easy to be deprotonated

104

Classification of Mechanism of CoA

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

Head Activation Mechanism(Acyl Group Transfer Mechanism), Cont’d

C

O

SR CoA

Nu..

C

O

SR Nu + CoAS

Good leaving group

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

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

• 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

C OH

O

O

CH3CH C

O

S CoA..

CH3CH C S CoA

C

O

O O


Acyl-CoA -carbanion

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


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


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

3. Siamese Twin Reaction(Acyl Transfer and Enolization Mechanism),

Cont’d

C

O

S CoAC

H

H

H

B:

C

O

S CoAC

H

H

B H + -

Delocalization of the negative charge

Acyl-SCoA (Thioester)

115


Cont’d

C

O

S CoAC

H

H

BH +C

O

S CoAC

H

H

Carbanion enolate Transition state intermediate

116


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


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

C

O

C

H

H

H

B:

C

O

C

H

H

B H O O


Cont’d

Acetate anion Unstabilized transition state

+ -

119


Cont’d

C

O

C

H

H

BH +C

O

C

H

H

O O

Acetate enolateKinetically unfavorable intermediate

120

4. Addition Reaction

• Reactions involving additions to CoA group• Example: Enoyl-CoA hydratase

121

5. Acyl Group Interchange Reaction

• Reactions involving acyl group interchange• Example: Acetoacetyl-CoA transferase

122

Mechanism of Succinyl-CoA Synthetase

(Succinyl Thiokinase )(Head Activation Mechanism)

123

Reaction of Succinyl-CoA Synthetase

G˚ = - 2.9 kJ/mol

124

Structure of Succinyl-CoA Synthetase

• The enzyme is an 22 heterodimer; the functional units is one pair

http://chemistry.gsu.edu/glactone/PDB/Proteins/Krebs/1scu.pdb

125

Mechanism of Succinyl-CoA Synthetase

(Head Activation Mechanism)

126

Mechanism of Succinyl-CoA Synthetase(Head Activation Mechanism)

HisC

SCoA

O

CC

SCoA

OH2 )( 2

COON

N

HPO O

O

OH

CH2)( 2

COO

OHis

N

N

H

PO

O

OH

BH

It is the displacement of CoA by Pi which generates another high energy compound, succinly-phosphate (phosphoester)

Pi

Succinyl-CoATetrahedral intermediate

Head activation

127


His

N

BH

B:

CoASH

C O

CH2)( 2

COO

OPO

O

OH

His

N

NH

C

CH2)( 2

COO

O

PO

O

OH

O

N

His removes the phosphoryl group with the concomitant generation of succinate and phosphohistidine

Succinlyl-Phosphat phosphohistidine

128


CH2

COO

COO

CH2

His

GDP

BH

N

N

PO

O

OHPO

O

OH

OGMphosphohistidine

Succinate

GDP

129


His

GTP

N

N

H

130

Mechanism of Citrate Synthtase

(Tail Activation Mechanism)

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

http://images.google.com/imgres?imgurl=http://chemistry.umeche.maine.edu/CHY431/1cts-1.gif&imgrefurl=http://chemistry.umeche.maine.edu/CHY431/Conformation2.html&h=367&w=435&sz=43&hl=en&start=95&um=1&tbnid=atwNf_flC-marM:&tbnh=106&tbnw=126&prev=/images%3Fq%3Dcitrate%2Bsynthase%26start%3D92%26ndsp%3D18%26svnum%3D10%26um%3D1%26hl%3Den%26safe%3Dactive%26sa%3DN

132

Reaction of Citrate Synthase

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

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

http://upload.wikimedia.org/wikipedia/en/a/a8/Citrate_Synthase_Active_Site.jpg

135

CS open State

http://upload.wikimedia.org/wikipedia/en/4/4c/Citrate_Synthase_%28open_form%29.png

136

CS Closed State

137

Reaction of Citrate Synthase

138

Reaction of CS, Cont’d

OAA Aceyl-CoA CoA Citrate

Ordered Mechanism

E E-OAA E-OAA-Acyl-CoA E-citrateE-citryl-CoA E

139

CS Stereochemistry

140

Stereochemistry of the CS Reaction

141

Stereochemistry of the CS Reaction, Cont’d

142


143


144

Mechanism of Citrate Synthase (Tail Activation Mechanism)

145

Mechanism of CS

N

N

His

C

SCoA

O

C H

H

H

N

N

His-320

C

OO 274

Asp 375

COO

C O

CH2

COO

H H

H N

N

HC

SCoA

H

H

HN

N

C

OO

COO

C O

CH2

COO

H H C

BH+

OH B

OAA

Deprotonation of -H+

CoAEnol intermediate

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

N

N

HC

SCoA

H

H

HN

N

C

OO

COO

CO

CH2

COO

H H C

OH B

BH+

N

NC

SCoA

H

H

HN

N

C

OO

COO

CO

CH2

COO

H H C

OH

BH+

H

BH+


Enol Carbanion intermediate

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


149

N

N

C

SCoA

HN

N

C

OO

COOCO

CH2

COO

O

H

B:

H

H

H2

CH2O

O

H

H

N

N

C

SCoA

HN

N

C

OO

COOCO

CH2

COO

O

H

BH+

H

H2

CO

H

H


Citryl-CoA (Thioester) intermediate

Hydroxlysis of citryl-CoA intermediate


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


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


152

N

N

N

N

C

OO

H

BH+

H

C

HSCoA

COOCO

CH2

COO

O

H

H2

O

H

C


Citrate

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


Documents

1 Part 5 Coenzyme-Dependent Enzyme Mechanisms Professor A. S. Alhomida Disclaimer The texts, tables and images contained in this course presentation are