1 part 3 coenzymes-dependent enzyme mechanism professor a. s. alhomida disclaimer the texts, tables...

1

Part 3 Coenzymes-Dependent Enzyme

MechanismProfessor A. S. Alhomida

Part 3 Coenzymes-Dependent Enzyme

MechanismProfessor 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

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Thiamine

4

Thiamine

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

Pyrimidine Thiazole

5

• Thiamin is derived from a substituted pyrimidine and a thiazole which are coupled by a methylene bridge. Thiamin is rapidly converted to its active form, thiamin pyrophosphate, TPP, in the brain and liver by a specific enzymes, thiamin diphosphotransferase. TPP is necessary as a cofactor for the pyruvate and a-ketoglutarate dehydrogenase catalyzed reactions as well as the transketolase catalyzed reactions of the pentose phosphate pathway. A deficiency in thiamin intake leads to a severely reduced capacity of cells to generate energy as a result of its role in these reactions. The dietary requirement for thiamin is proportional to the caloric intake of the diet and ranges from 1.0 - 1.5 mg/day for normal adults. If the carbohydrate content of the diet is excessive then an in thiamin intake will be required.

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Conversion of Thiamine 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

Thiamine Thiamine pyrophosphate (TPP)

TPP synthetase

7

• 3-D structure

Structure of TPP, Cont’d

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Structure of TPP, Cont’d

9

Thiamin Biosynthesis

• The thiamin thiazole in Bacillus subtilis is biosynthesized from 1-deoxy-D-xylulose-5-phosphate (1, DXP), glycine imine 11 and a sulfur carrier protein thiocarboxylate (4, ThiS–COS–) as outlined

• In this mechanism, DXP forms an imine with lysine 96 of the thiazole synthase (ThiG), which then tautomerizes to 3

10

• Addition of ThiS-thiocarboxylate 4, followed by an acyl shift and loss of water gives 7

• Tautomerization of 7 followed by elimination of ThiS–COO– 10 gives 9 which then adds to the glycine imine 11, formed by the oxidation of glycine, to give 12. Transimination followed by decarboxylation completes the thiazole formation

Thiamin Biosynthesis, Cont’d

11

• This mechanism is supported by isotope exchange experiments, intermediate trapping, the demonstration of oxygen transfer from DXP to ThiS–COO– and the structural characterization of thiazole synthase (ThiG) complexed to the sulfur carrier protein

• This thiazole biosynthesis is different from any of the characterized chemical or biochemical routes to the thiazole heterocycle


12

• A related oxygen-sensitive thiazole biosynthesis in Escherichia coli that uses tyrosine instead of glycine has been reconstituted but not yet mechanistically characterized


13


14

TPP and TCA Cycle

• The mechanism is identical for both the conversion of pyruvate to acetyl CoA and the conversion of -KG to succinyl CoA

• In the reaction, the proton on C-2 of TPP dissociates to give a carbanion

15

TPP and TCA Cycle, Cont’d

• Nucleophilic addition by the carbanion to the carbonyl group of the -keto acid (i. e. pyruvate or -KG) followed by protonation forms an activated -hydroxyacid

• The hydroxy acid then undergoes decarboxylation

• The positively charged nitrogen of TPP serves as a critical electron sink during the decarboxylation step and contributes to the resonance stabilization of the hydroxyalkyl-TPP decarboxylation product

16

TPP and TCA Cycle, Cont’d

• The hydroxyalkyl group is transferred by other proteins in the complex to CoA to produce acetyl CoA from pyruvate or succinyl CoA from -KG

17

TPP and Pentose Phosphate Pathway (PPP)

• PPP harvests energy from fuel molecules and stores it in the form of NADPH

• NADPH is an important electron donor in reductive biosynthesis

• PPP also produces 5-carbon sugars such as ribose which is used in the synthesis of DNA and RNA

18

TPP and PPP, Cont’d

• TPP is the coenzyme for the transketolase• Transketolase transfers a 2-carbon unit from

an -ketose• 2-carbon unit from the 5-carbon -ketose

xylulose 5-phosphate is transferred to the 4-carbon aldose erythrose 4-phosphate to make the 6 carbon -ketose fructose 6-phosphate

• GAP results from the 3-carbon fragment that is cleaved from xylulose 5-phosphate

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• Carbanion at C-2 of TPP is first produced that attacks the carbonyl carbon of the -ketose to give an addition product

• After deprotonation of OH- group an aldose (in this case GAP) is released and an activated glycoaldehyde bound to TPP is produced

• The thiazole nitrogen serves as electron sink in the reaction and contributes to resonance stabilization of the resulting product (activated glycoadehyde)


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• This glycoaldehyde is said to be activated because it is also a carbanion and readily undergoes nucleophilic addition to the carbonyl group of an aldose (erythrose 4-phosphate here)

• Following another deprotonation the nascent -ketose is released from TPP


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Neurological Function of TPP

• It is evident from the neurological disorders caused by thiamine deficiency that this vitamin plays a vital role in nerve function

• It is unclear, however, just what that role is• Thiamine is found in both the nerves and

brain• The concentration of thiamine in the brain

seems to be resistant to changes dietary concentration

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Neurological Function of TPP

• Electrical or chemical (e.g., acetylcholine) stimulation of nerves results in the release of thiamine monophosphate (TMP) and free thiamine into the medium with accompanying decrease of cellular TPP and thiamine triphosphate (TTP)

• This observation suggest that thiamine has a role in the nervous system independent of its coenzyme roles

• One theory is that TTP is involved with nerve impulses via the Na+ and K+ gradient

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Thiamine Deficient Diseases(Beri-beri)

• Thiamine deficiency usually causes weight loss, cardiac abnormalities, and neuromuscular disorders

• The classic thiamine deficiency syndrome in humans is beri-beri (sometimes called Kakke)

• Thiamine is abundant in whole grains, usually in the scutellum (the thin covering of the starchy interior endosperm), but is scarce in the endosperm

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Thiamine Deficient Diseases(Beri-beri), Cont’d

• Unfortunately beri-beri is still common in parts of southeast Asia where polished rice is a staple and thiamine enrichment programs are not fully in place

• Beri-beri is characterized by anorexia (loss of appetite) with subsequent weight loss, enlargement of the heart, and neuromuscular symptoms such as paresthesia (spontaneous sensations, such as itching, burning, etc), muscle weakness, lassitude (weariness, general weakness), and foot and wrist droop

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• There are three main types of beri-beri: (1) Dry (also neuritic, paraplegic, and pernicious) beri-beri; (2) Wet (also edematous or cardiac) beri-beri; (3) Infantile (also acute) beri-beri

• Dry beri-beri usually inflicts older adults and affects mainly the peripheral nerves with

• It is characterized by atrophy (wasting away) and peripheral neuritis (inflammation of nerves) of the legs and paraplegia (paralysis of the lower extremities)

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• In contrast wet beri-beri displays substantial cardiac involvement especially tachycardia (rapid heart beat) in addition to peripheral neuropathy

• Edema progresses from the feet upwards to the heart causing congestive heart failure in severe cases

• Infantile beri-beri is usually seen in breast-feeding infants whose mothers are thiamine deficient (but not necessarily showing signs of beri-beri)

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• These infants are usually anoretic and often have trouble keeping the milk down

• Once the disease begins it moves rapidly causing heart failure in a matter of hours

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Wet Beri-Beri

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TPP and Wernicke-Korsakoff Syndrome

• It is the thiamine deficient disease seen most often in the Western hemisphere

• It mainly affects alcoholics due to three reasons: (1) Diets of alcoholics are usually poor

(2) Diets rich in carbohydrates (e.g., alcohol or rice) increase the metabolic demands of thiamine

(3) Alcohol inhibits intestinal ATPase which is involved in the uptake of thiamine

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TPP and Wernicke-Korsakoff Syndrome

• Two observations suggest a genetic involvement with Wernicke-Korsakoff Syndrome:(1) It is much higher in among Europeans than non-

Europeans

(2) Transketolase from these patients binds TPP 10 time less strongly than normal transketolase

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Decarboxylatoion Reactions

34

• 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

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Decarboxylation Reaction, Cont’d

C C

O

O

R1

R3

R2

CR1

R3

R2

+

O

C

O

Carboxylic acid Carbanion (unstable)

CO2 (stable)

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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

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• -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


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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

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C C

O

O

+

O

C

O

C

H

H

CH3 C

O

C

H

H

CH3

H

H


Not -ketoacid

No Electron sink

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• 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


41

• 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


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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

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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

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• 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


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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

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• 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


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• -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


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• 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


49

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


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Decarboxylation of -Keto Acid

51

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

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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

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Biochemical Functions of TPP

54


• Reaction with the -keto acid generates a heterocyclic enol and carbon dioxide

• Although the enol is relatively stable it retains its activity because it has lost fully aromatic character

• The enol reacts as a nucleophile with -carbonyl compounds

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-Keto acidTPP

TPP carbanion

BH+

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• Transfer of the acetyl group restores the aromatic thiazolium system

• Note that the acetyl group is formally transferred as an anion with its negative charge on the carbon of the carbonyl group

• This is reversed polarity and constitutes an Umpolung

Biochemical Functions of TPP, Cont’d

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• Reaction with the -keto acid generates a heterocyclic enol and carbon dioxide

• Although the enol is relatively stable it retains its activity because it has lost fully aromatic character

• In TPP the OH group of thiamine is replaced by a diphosphate ester group

58


Heterocycle enolTPP carbanion -Keto acidHE-TPP

Electron sink

59


• The reaction site of TPP is C-2 of the thiazole ring

• Thiazolium ring is responsible for the enzymatic catalysis carried out by TPP due to chemical prosperities:(1) The acidity of the proton attached to C-2

(2) The presence of a C = N (double bond) that can act as an electron sink for decarboxylation

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• The proton on this carbon is rather acidic because of the adjacent positively charged quaternary nitrogen atom which electrostatically stabilizes the carbanion intermediate formed when the proton dissociates

• The pKa value of C-2 is near 10 (dipolar carbanion)

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• TPP-C-2 carbanion appears unusually stable due to electrostatic interaction with the cationic nitrogen and also d-p orbital overlap of the negative charge with the adjacent sulfur atom

• TPP-carbanion has an efficient electron sink in form of -iminium ion in -relation to the carboxylate group to be decarboxylated as CO2

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• This intermediate provides a low-energy path to facilitate the decarboxylation reaction of -keto acid

• When this proton dissociates a carbanion is formed which readily undergoes nucleophilic addition to-carbonyl groups

• The carbanion readily adds to carbonly groups, and the thiazolium ring acts as electron sink that stabilizes the negative charge that is transferred to the ring

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NS

RCH3

R` NO

RCH3

R`

HH

N N

RCH3

R`

H

H

Thiazolium Oxazolium Imidazolium

64

Comparison Studies

• 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 and 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

•

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• 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

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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

67


• Both of which cleave a C-C bond adjacent to a carbonyl group releasing either carbon dioxide or an aldehyde

• The resulting product is then transferred to an acceptor molecule

• -Keto acid dehydrogenases decarboxylate -keto acids

• The decarboxylation product is then transferred to CoA

68


• Transketolases cleaves the C-C bond adjacent to the carbonyl group of an -ketosugar to give an activated glycoaldehyde

• The glycoaldehyde is then combined with an aldose to give a new ketose

• All known TPP dependent enzymes also require a divalent cation, commonly Mg2+

69

Mechanism of Pyruvate Dehydrogenase (PDH)

Complex

70

Reaction of PDH Complex

71

Reaction of PDH Complex, Cont’d

72

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

73

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

74

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)

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Structure of PDH Complex, Cont’d

76

• 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


77

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

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• 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


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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

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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

81

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

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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

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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

84

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


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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


86

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


87

Mechanism of PDH Complex

88

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

89

Formation of TPP-carbanion, Cont’d

N

N HH

H3C

CH2 NS

RCH3

Glu

CO

O

Electron sink to stabilize the negative charge

90


TPP carbanion

Pyruvate

Pyruvate decarboxylase

C

CH3

R2

R1

N

S

CH3

C O

OC

O

BH

91


• The mechanism is identical for both the conversion of pyruvate to acetyl CoA and the conversion of -KG to succinyl CoA

• In the reaction, the proton on C-2 of TPP dissociates to give a carbanion

• Nucleophilic addition by the carbanion to the carbonyl group of the -keto acid (i. e. pyruvate or -KG) followed by protonation forms an activated -hydroxy acid

92

Mechanism of PDH complex

• The first step of this reaction, decarboxylation of pyruvate and transfer of the acetyl group to lipoamide, depends on accumulation of negative charge on the carbonyl carbon of pyruvate

• This is facilitated by the quaternary nitrogen on the thiazolium group of TPP

93

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

94

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

95

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(

96

Mechanism of PDH complex, Cont’d

• The cationic imine nitrogen plays three distinct and important roles in TPP-catalyzed reactions:(1) It provides electrostatic stabilization of the

carbanion formed upon removal of the C-2 proton (The sp2 hybridization and the availability of vacant d orbitals on the adjacent sulfur probably also facilitate proton removal at C-2)

(2) TPP nucleophilically attack on pyruvate leads to decarboxylation

(3) TPP cationic imine nitrogen can act as an effective electron sink to stabilize the negative charge that must develop on the carbon that has been attacked

97

• The hydroxy acid then undergoes decarboxylation

• The positively charged nitrogen of TPP serves as a critical electron sink during the decarboxylation step and contributes to the resonance stabilization of the hydroxyalkyl-TPP decarboxylation product

• The hydroxyalkyl group is transferred by other proteins in the complex to CoA to produce acetyl CoA from pyruvate or succinyl CoA from -KG


98

• This stabilization takes place by resonance interaction through the double bond to the nitrogen atom

• This resonance-stabilized intermediate can be protonated to give hydroxyethyl-TPP

• This well-characterized intermediate was once thought to be so unstable that it could not be synthesized or isolated


99

• However, its synthesis and isolation are actually routine (In fact, a substantial amount of the TPP in living things exists as the hydroxyethyl form)

• The reaction of hydroxyethyl-TPP with the oxidized form of lipoamide yields the high-energy thiolester of reduced lipoamide and results in oxidation of the hydroxyl-carbon of the two-carbon substrate unit


100

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


101

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

102

• This is followed by nucleophilic attack by CoA on the carbonyl-carbon

• The result is transfer of the acetyl group from lipoamide to CoA

• The subsequent oxidation of lipoamide is catalyzed by the FAD–dependent dihydrolipoyl dehydrogenase and NAD+ is reduced


103

Regulation of the PDH Complex

104


• The reactions of the PDH complex serves to interconnect the metabolic pathways of glycolysis, GNG and fatty acid synthesis to the TCA cycle

• As a consequence, the activity of the PDH complex is highly regulated by a variety of allosteric effectors and by covalent modification

105

Regulation of the PDH Complex, Cont’d

• The importance of the PDH complex to the maintenance of homeostasis is evident from the fact that although diseases associated with deficiencies of the PDH complex have been observed, affected individuals often do not survive to maturity

• Since the energy metabolism of highly aerobic tissues such as the brain is dependent on normal conversion of pyruvate to acetyl-CoA

106


• Aerobic tissues are most sensitive to deficiencies in components of the PDH complex

• Most genetic diseases associated with PDH complex deficiency are due to mutations in PDH

• The main pathologic result of such mutations is moderate to severe cerebral lactic acidosis and encephalopathies.

107


108

• NADH and acetyl-CoA, are negative allosteric effectors on PDH-a, the non-phosphorylated, active form of PDH

• These effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the PDH complex

• NADH and acetyl-CoA are powerful positive effectors on PDH kinase, the enzyme that inactivates PDH by converting it to the phosphorylated PDH-b form


109

• Since NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDH kinase activity, reinforcing down-regulation of PDH activity in energy-rich cells

• Note, however, that pyruvate is a potent negative effector on PDH kinase, with the result that when pyruvate levels rise, PDH-a will be favored even with high levels of NADH and acetyl-CoA


110

• Concentrations of pyruvate which maintain PDH in the active form (PDH-a) are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA

• With large amounts of pyruvate in cells having high energy charge and high NADH, pyruvate carbon will be directed to the 2 main storage forms of carbon (glycogen via GNG and fat production via fatty acid synthesis)


111

• Although the regulation of PDH-b phosphatase is not well understood, it is quite likely regulated to maximize pyruvate oxidation under energy-poor conditions and to minimize PDH activity under energy-rich conditions


112

Structure of Dihydrolipoly Transacelyase

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

113

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

Structure of Dihydrolipoly Transacelyase, Cont’d

114

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

115

• 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

116

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

117

• 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

118

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+

119

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

120

Transketolase

121

• The pentose phosphate pathway (PPP) harvests energy from fuel molecules and stores it in the form of NADPH

• NADPH is an important electron donor in reductive biosynthesis

• The PPP also produces 5-carbon sugars such as ribose which is used in the synthesis of DNA and RNA

122

• TPP is the coenzyme for the enzyme transketolase

• Transketolase transfers a 2-carbon unit from an -ketose (a sugar with a carbonyl group at position 2) to an aldose

• 2-carbon unit from the 5-carbon -ketose xylulose 5-phosphate is transferred to the 4-carbon aldose erythrose 4-phosphate to make the 6 carbon -ketose fructose 6-phosphate

123

• Glyceraldehyde 3-phosphate results from the 3-carbon fragment that is cleaved from xylulose 5-phosphate

• Carbanion at C-2 of TPP is first produced• This carbanion attacks the carbonyl carbon of

the -ketose to give an addition product• After deprotonation of the appropriate

hydroxyl group an aldose (in this case glyceraldehyde 3-phosphate) is released and an activated glycoaldehyde bound to TPP is produced

124

• The thiazole nitrogen serves as electron sink in the reaction and contributes to resonance stabilization of the resulting product (activated glycoadehyde)

• Following another deprotonation the nascent -ketose is released from TPP

125

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

126

Structure of Transketolase

3- D Structure of yeast

127

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

128

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

129

Mechanism of Transketolase

130

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

131

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

132

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

133

Coenzyme A

134

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

135

Biosynthesis of CoASH

138

Biosynthesis of CoASH, Cont’d

• Coenzyme A (CoASH or CoA) is synthesized in a five-step process from pantothenate:(1) Pantothenate is phosphorylated to 4'-

phosphopantothenate by the enzyme pantothenate kinase

(2) A cysteine is added to 4'-phosphopantothenate by the enzyme phosphopantothenoylcysteine synthetase to form 4'-phospho-N-pantothenoylcysteine (PPC)

139

(3) PPC is decarboxylated to 4'-phosphopantetheine by phosphopantothenoylcysteine decarboxylase

(4) 4'-phosphopantetheine is adenylylated to form dephospho-CoA by the enzyme phosphopantetheine adenylyl transerase

(5) Finally, dephospho-CoA is phosphorylated using ATP to coenzyme A by the enzyme dephospho-coA kinase

Biosynthesis of CoASH, Cont’d

140

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'

141

• CoA itself is a complex and highly polar molecule, consisting of adenosine 3',5'-diphosphate linked to 4-phosphopantethenic acid (vitamin B5) and thence to β-mercaptoethylamine, which is directly involved in acyl transfer reactions

• The adenosine 3’,5’-diphosphate moiety functions as a recognition site, increasing the affinity of CoA binding to enzymes

Function of CoASH, Cont’d

142

• Not only is CoA associated intimately with most reactions of fatty acids, but it is also a key molecule in the catabolism of carbohydrates via the TCA cycle in which acetyl-CoA is a major end-product

• The genes encoding the enzymes for coA biosynthesis have been identified and the structures of many proteins in the pathway have been determined


143

• Although there are substantial sequence differences between prokaryotes and eukaryotes, coA is assembled in five steps from pantothenic acid in essentially the same way in both groups

• However, pantothenic acid per se can only be synthesized by microorganisms and plants and must be acquired from the diet by animals


144

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 may types of organism is transfer of the fatty acyl group from ACP to CoA

145

Structure of CoASH

146

Structure of CoASH

http://upload.wikimedia.org/wikipedia/commons/c/cf/Coenzyme-A-3D-balls.png

147

Deficiency of Pantothenic

• 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

148

Biochemical Features of Coenzyme A

• Nature's ester enolate equivalent is CoA• It carries a free thiol group to which carboxylic

acid residues are transferred• Thioesters are very reactive intermediates• They are both activated towards nucleophilic

attack (electrophilicity of the carbonyl group) and abstraction of a proton (acidity of the -proton)

149


Good leaving group

Enolization reactionAcyl transfer reaction

150

• The Claisen reaction between acetyl-CoA and malonyl-CoA illustrates how -keto esters are built up by nature using the enolate derived from acetyl-CoA as nucleophile


151


152

• How do living systems synthesize the amide bonds found in proteins, or the ester functional groups found in lipids, oils and other natural products?

• Carboxylic acid is not suitable for transferring acyl goup at physiological conditions and should be activated

• Many reactions in metabolism involve acyl-group transfer or enolization of carboxylic acid that exit as unactivated carboxylate anion at the physiological pH

Activation of Carboxylate Anion

153

• The general mechanism is to make an activated acyl derivative containing a good leaving group, and then to carry out an acyl transfer reaction

• Activation and transfer of acyl groups is a common mechanism found in proteins, fatty acis biosynthesis and polyketide natural products biosynthesis

Activation of Carboxylate Anion

154

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

155

• The predominant mechanism by which carboxylic acids are activated for acyl transfer and enolization is esterification with the thiol group of CoASH

• CoASH is well suited to carry out acyl reactions, since thiols are inherently more nucleophilc than alcohols or amines

• Thiols are also better leaving groups (pKa 8-9), which explains why the hydrolysis of thioesters under basic conditions is more rapid that oxyester hydrolysis

Activation of Carboxylate Anion, Cont’d

156


Thioester (Acyl-CoA)

Good leaving group

Activation of Carboxylate Anion by CoASH

CR

O

O

+ SCoA

H

B:

H

CR SCoA

BHH

O

O

CR SCoA

OH2O

B:

157

Acyl Transfer Using Acyl-CoA

Good leaving group


AcylesterAcceptor for acyl group

CR SCoA

O

+ YR` H

B:

CR SCoA

O

YR` BH

CR

O

YR`

+ CoASH

Y = alcohol, amine, carbanion, or thiol group

Thioester

158

• Nucleophilic attack to the neutral activated acyl group is a favored process and CoA is a good leaving group from tetrahedral intermediate

159

Thioester vs Oxyester

• 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 thioester in

condensation (Claisen) reactions because the carbonyl carbon atom has more positive character than the carbonly in the corresponding oxyester

160

Thioester vs Oxyester, Cont’d

• 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

161

• 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....

162

• 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


163


C

O

R....SC

H

H

H C

O

R....OC

H

H

H

ThioesterOxyesterMore positive

charge

Less positive charge

Easy to be deprotonated

Not easy to be deprotonated

164

• Activation and Coupling• Formation of an amide from a carboxylic acid and an amine

often results in overall loss of free energy however there is a high activation energy to be overcome. To make the synthesis at all viable this energy must be lowered. This is achieved either by catalysis or by formation of carboxylic acid derivatives (denoted RCOX), effectively starting at a higher energy on the free energy reaction pathway (Figure). The nature of the leaving group 'X' governs the value of the activation energy. It would thus seem desirable to form amino acid derivatives with a strongly electron withdrawing 'X', making the carbonyl carbon more prone to nucleophilic attack and thereby achieving high reaction rates at ambient temperatures.

166

• Initially the azide and chloride groups were proposed as the 'X' substituent. While the azide proved to be almost entirely suited to its task, the acid chloride suffers from the problem of being "over-activated". Due to the ease of elimination of the chloride ion, the carbonyl is prone to attack from even weak nucleophiles. They are thus prone to hydrolysis and cannot be conveniently stored.

• Another more subtle side reaction is the loss of chiral integrity at the -centre of the activated amino acid. This can occur through two mechanisms:

167

• 1) Direct abstraction of the a-proton.

• 2) Formation of an oxazolone (also known as an azlactone), increasing the acidity of the a-proton.

• Both these situations result in the formation of a planar carbanion with reprotonation possible from both faces (Figure).

168

• Carboxyl Protecting Groups.• Protection of the carboxyl group is required for three main reasons:

•

• 1. To enhance the solubility of the peptide in organic solvents.

• 2. To avoid anhydride formation in the presence of activated amino acids.

• 3. To allow unambiguous activation in the presence of a coupling reagent (only the unprotected carboxyl group can be activated).

•

169

Pyridoxal Phosphate (PLP)

170

Pyridoxine in Foods

171

Forms of pyridoxal-5`-phosphate

(a) Pyridoxine (vitamin B6), (b) Pyridoxal-5`-phosphate (PLP), (c) Pyridoxamine-5`-phosphate (PMP) and (d) The Schiff base that forms between PLP and an enzyme -amino group

172

• The biologically active form of vitamin B6 is pyridoxal-5-phosphate (PLP), a coenzyme that exists under physiological conditions in two tautomeric forms

• PLP participates in the catalysis of a wide variety of reactions involving amino acids, including transaminations, - and -decarboxylations, - and -eliminations, racemizations, and aldol reactions

Pyridoxine (vitamin B6)

173

• Note that these reactions include cleavage of any of the bonds to the amino acid -carbon, as well as several bonds in the side chain

• The remarkably versatile chemistry of PLP is due to its ability to:– (a) Form stable Schiff base (aldimine) adducts

with -amino groups of amino acids– (b) Act as an effective electron sink to stabilize

carbanion intermediates

Pyridoxine (vitamin B6)

174

Vitamin B6 (Pyridoxine)

• Vitamin (B6), Pyridoxine, 2-methyl-3-hydroxy-4,5-bis(hydroxy-methyl)pyridine, is essential for protein metabolism, and for the formation of hemoglobin

• Pyridoxine is needed by rats to cure dermatitis developed on a Vitamin B -free diet supplemented by thiamine and riboflavin.

• Its absence from diet is also associated with anemia

• It is needed also by certain bacteria

175

• The related compounds:– Pyridoxamine– Pyridoxal

• They posse vitamin B6 activity and are much more active than pyridoxine

• Good sources of Vitamin B6 are rice husks, maize, wheat germ, yeast and other sources of vitamin B

• Food contains three natural forms of vitamin B6: pyridoxine, pyridoxamine, and pyridoxal

Vitamin B6 (Pyridoxine), Cont’d

176

• It exists in different forms; one of those forms, pyridoxal 5'-phosphate (PLP), serves a cofactor in many enzyme reactions, including the transsulfuration pathway, in which homocysteine is converted to cystathionine and then to cysteine


177

• Pyridoxine is a water-soluble vitamin• The body metabolizes this to pyridoxal

phosphate (PLP), which the active cofactor• PLP is involved in many different types of

reactions involving amino acids• Reactions may involve the , , or carbon

of the amino acid


178

• Reactions at the -carbon include: 1. Transamination reactions

2. Aldol cleavages

3. Deaminations

4. Decarboxylations

5. Racemizations

• Reactions at the -carbon include:6. Eliminations

7. Replacements


179

• Reactions at the -carbon include: 8. Eliminations

9. Replacements


180

• The commercial vitamin form, pyridoxine hydrochloride, has the hydrochloride added for stability and increased shelf life. That form is artificial but is well utilized by most individuals

• However, the body cannot use pyridoxine directly

• Two metabolic steps are needed:


181

• First, the pyridoxine must be phosphorylated, that is, phosphate is added to the ring-structure of the molecule

• Pyridoxine, pyridoxal, and pyridoxamine are all well-absorbed through the mucosa of the small intestine

• Inside cells, all these forms are phosphorylated using the enzyme pyridoxal kinase


182

• Magnesium is needed to activate kinase enzymes-enzymes that phosphorylate

• However, there is published experimental work, showing in vitro, that this particular phosphorylating enzyme in human brain tissue has higher affinity for zinc and higher activity with zinc than with magnesium


183

• After phosphorylation, if the cell started with pyridoxal, the biochemistry is completed

• The pyridoxal 5-phosphate (PLP) coenzyme is ready to go to work

• The phosphorylating kinase prefers pyridoxal over pyridoxine

• The enzyme phosphorylates pyridoxal faster than it phosphorylates pyridoxine


184

• With pyridoxine, the vegetable source form of B6, this phosphorylation produces pyridoxine phosphate

• Then next, the pyridoxine phosphate has to be oxidized by an oxidase enzyme that is assisted by vitamin B2, riboflavin, as FAD


185

• PLP functions as a coenzyme in enzymes involved in transamination reactions required for the synthesis and catabolism of the amino acids as well as in glycogenolysis as a coenzyme for glycogen phosphorylase


186

Metabolism of Pyridoxine

From Diet

Pyridoxine

Pyridoxine Kinase

Pyridoxine phosphate

N

O

CH3

HO

ATP ADP

CH2OH

HN

O

CH3

O

CH2OH

H

P

O

O

HO

187

Pyridoxine phosphate Pyridoxal 5- phosphate

(PLP)

Oxidase

Metabolism of Pyridoxine, Cont’d

N

O

CH3

O

CH2OH

H

P

O

O

HO

FADFADH2

N

O

CH3

O

CHO

H

P

O

O

HO

188

Pyridoxal 5- phosphate

(PLP)

Pyridoxamine phosphate

(PMP)

Transaminase


N

O

CH3

O

CHO

P

O

O

HO

Asp OAA

N

O

CH3

O

CH2NH2

P

O

O

HO

H H

189

Phosphatase

Dehyrogenase

Excreted in urine


PLPPyridoxal

4-Pyridoxic acid

N

O

CH3

O

CHO

P

O

O

HON

O

CH3

O

CHO

H

H2O Pi

H H

N

O

CH3

O

COO

H

H

NAD

NADH

190

Tautomeric Forms of Pyridoxal-5-Phosphate (PLP)

191

• The seven classes of reactions catalyzed by pyridoxal-5-phosphate

192

Features of PLP

• The Schiff base formed by PLP and acts as an electron sink to stabilize the carbanion

• All PLP-dependent enzymes, PLP in the absence of substrate is bound in a Schiff base linkage with the NH2 group of an active site of Lys (Internal Schiff base)

• PLP-dependent enzymes, substrate provides the amine group whereas the enzyme provides the carbonly group

193

• One key to PLP chemistry is the protonation of the Schiff base, which is stabilized by H bonding to the ring oxygen, increasing the acidity of the C proton (as shown in Rxn 3)

• The carbanion formed by loss of the C proton is stabilized by electron delocalization into the pyridinium ring, with the positively charged ring nitrogen acting as an electron sink

Features of PLP, Cont’d

194

• Another important intermediate is formed by protonation of the aldehyde carbon of PLP (as shown in Rxn 5)

• This produces a new substrate-PLP Schiff base, which plays a role in transamination reactions and increases the acidity of the proton at C, a feature important in elimination reactions


195

• The stereochimistry of the amino acid formed is determined by the direction from which the H+ is added to the quinonoid intermediate (VIII) which determined

• Rearrangement to a Schiff base with the arriving substrate is a transaldiminization reaction


196

Pyridoxal-5-phosphate (PLP) forms stable Schiff base adducts with amino acids and acts as an effective electron sink to stabilize a variety of reaction intermediates

EnzEnz

Protonation of Schiff base

Protonation of aldehyde carbon

Enz

1 part 3 coenzymes-dependent enzyme mechanism professor a. s. alhomida disclaimer the texts, tables...

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