1 part 2 coenzymes-dependent enzyme mechanism professor a. s. alhomida disclaimer the texts, tables...

1

Part 2 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 UniversityCollege of Science

Department of Biochemistry

3

Experimental Evidences for Stereochemistry Hydride Transfer is Stereospecific

4

NR

HCONH2

+ CCHH

HOH

D

D

yeastAD

NR

CONH2

DH

CCHH

HD

O+

NR

CONH2

DH

CCHH

HH

O+

yeastAD

NR

HCONH2

+ CCHH

HOH

H

D

*

CCHH

HOH

H

DCCH

H

HO

H

DSO

O

HOCCH

H

HOH

D

H* stereochemistry

inverted

CCHH

HOH

D

H+N

R

HCONH2 yeast

AD

NR

CONH2

HHCCH

H

HD

O+

*

*

50: 50

100%

5

Stereospecificity of ADH

• When the redox transformation involves only hydrogens, one cannot distinguish the stereochemical course

• However, if one uses deuterium labeling, one discovers that:

6

• The pro-R hydrogen of ethanol is removed • The hydrogen is transferred to the re face of

NAD+ (50 : 50)• If the reaction is run in reverse, the pro-R H of

NADH is transferred to the re face of acetaldehyde (100%)


7

• The implication of this stereospecificity is that when ethanol and NAD+ are bound to the enzyme, their binding sites must orient them so that the pro-R H of ethanol is directed toward the re face of the NAD+

• Therefore, the enzyme must bind at least two of the groups attached to the prochiral center, leaving the orientation of the NAD+ ring to distinguish between the two hydrogens


8

Structure of Active site of ADH

• The active site contains Zn bound to two Cys and one His

• The Zn ion binds the acetaldehyde through its oxygen atom to polarize it so that it more easily accepts a hydride ion (light blue) from NADH

9

ADH with bound NAD+

10

ADH with bound NAD+ and Trifluoroenanol (TFE)

• The zinc (orange) is coordinated to His-67, Cys-174, Ser-48, and a water molecule (not visible)

• Just above the Zn is the hydride acceptor ring of NAD+

• The alcohol binds to the Zn replacing the water molecule, and thus must lie between the Zn and the pyridinium ring

• Leu-57, Phe-93 (mutated to Trp in this case), and His-51 form the rest of the binding pocket

11

Conformation of the nicotinamide cofactor determines if thepro-R or the pro-S hydrogen is transferred from the cofactor

ORO

HO OH

N

O

H2N

HR

HS

no free rotation

Syn

ORO

HO OH

N

HS

HRCONH2

Anti

12

Conformation of NAD+ cofactors

NAD+ from Lactate Dehydrogenase (Pro-R specific)

NAD+ from Glyceraldehyde-3-phosphate Dehydrogenase (Pro-S specific)

13

Stereospecificity at C-4 for some NAD-Dependent DH

Enzyme Nucleotide C-4 pro H ProductADH NAD HR Acetaldehyde

UDP-glucose DH

NAD HS UDP-glucuronate

LDH NAD HR Pyruvate

MDH NAD HR OAA

ICDH(Cyt) NADP HR a-KG

ICDH(Mit) NAD HR a-KG

GAPDH NAD HS 1,3-BPG

Glu DH NADP, NAD HS a-KG, NH4+

14

Mechanism of Alcohol Dehydrogenase

15

Mechanism of Alcohol Dehydrogenase

Zn2+ Ser

O

H

N

NH

OH

CH

H

CH3

B:

N

R

NH2

O

His

NAD+

Ethanol

+

Cys-174

Cys-46 His-67

-48

-61

Electron sink (Stored 2 electrons and one H+). Source & Where?

The Zn2+ increases the acidity of the alcohol, but is not involved in the redox reaction

16

H2O

CH3C

Alcohol Dehydrogeanse

Zn2+ Ser

OH

OH

H

N

N

H

His

O

H

NADH

+N

NH2

R

HH

..

O

Acetaldehyde

BH+

17

Glyceraldehyde 3-phosphate Dehydrogenase

18


• GAPDH is one of the key enzymes for glycolysis, reversibly catalyzes the first glycolytic reaction to involve oxidation-reduction

• It converts the glyceraldehyde-3-phosphate (G3P) into the high energy phosphate compound, 1,3 bisphosphoglycerate (BPG), using NAD+ as a cofactor

• BPG reacts with ADP to from ATP by phosphoglcerate kinase

19


• In addition to its role in glycolysis, GAPDH is known to be involved in several other non-glycolytic functions as well (e.g. apoptosis, DNA repair and regulation of histone gene expression

20


• It contains an active site Cys, which helps explain how the enzyme can be inactivated with a stoichiometric amounts of iodoacetamide

• Both glycolytic and non-glycolytic functions are targeted for drug design

• It is one of the few examples in which the coupling of an oxidation (favorable Rxs) to phosphorylation (unfavorable Rxs) process

21


• It is a medium-sized tetrameric enzyme • It resembles several other tetrameric NAD-

dependent oxidoreductases, like LDH, ADH and MDL; all have characteristic structures in the NAD-binding region known as "Rossmann folds", after Michael Rossmann, who first characterized this class of enzymes structurally

http://www.csrri.iit.edu/~howard/biol403/structures/1GD1x500.jpg

22

Structure of GAPDH shows the tetramer subunits

23

GAPDH Reaction

G˚ = 6.3 kj/mol

24

Experimental Evidence for Pi Role

25

What is the role of Pi?

GAPDHNo product

1. If run with catalytic amount of enzyme and omit Pi from the mixture of the reaction, no product is formed

CH

OHC

CH2OP

H

O

O2

3

+ NAD+

3-PG

26

Experimental Evidence for Pi Role, Cont’d

2. If run with a relatively large amount of enzyme (so that enzyme-bound intermediates could be detected) and omit Pi from the mixture of the reaction, the results showed at time zero, a rapid increase in the absorbance was observed, that is, NADH was formed

3. However, the reaction stopped when all the enzyme-bound NAD+ has been reduced

27


4. At this point, only small amount of the available free NAD+ and substrate had been consumed

GAPDHC

H

OHC

CH2OP

H

O

3-PG

O2

3

+ NAD+

C

OHC

CH2OP

H

O

1, 3 bPG

O2

3

+ NADH

OPO32

28


5. When Pi was added, more NADH was rapidly formed

6. The amount of NADH formed was equivalent to the amount of Pi added and considerably larger than the amount of enzyme present

C

H

OHC

CH2OP

H

O

3-PG

O2

3

+ Pi

C

OHC

CH2OP

H

O

1, 3 bPG

O2

3

OPO32

NAD+NADH

PAPDH

29

Results of the Experimental Evidence for Pi Role

Time (min)

Abs

orba

nce

(340

nm

)

Add Pi

1st burst of NADH formation

2nd burst of NADH formation

30

GAPDH Reaction, (Cont’d)

The +ve on the NAD+ may help polarize the thioester to facilitate the attack by Pi

Oxidation step

Phosphorylation step

31

Structure of GAPDH shows the active site includes Cys, His residue adjacent to a bound NAD+

32


• A general base in the enzyme abstracts an H+ from Cys, which attacks the carbonyl C of the glyceraldehyde, forming a tetrahedral intermediate

• A hydride leaves from the former carbonyl C to NAD+ in an oxidation step

• Notice, this is a two electron oxidation reaction similar to seen in alcohol dehydrogenase

33

Glyceraldehyde 3-phosphate Dehydrogenase, Cont’d

• GAPDH reaction is the site of action of arsenate (AsO4

3-), an anion analogous to phosphate

• Arsenate is an effective substrate in this reaction, forming 1-arseno-3-phosphoglycerate (1-Ars-PG), but acyl arsenates are quite unstable and are rapidly hydrolyzed

34

• 1-Ars-PG breaks down to yield 3-PG

• The result is that glycolysis continues in the presence of Ars, but the molecule of ATP is not made because this step has been bypassed

Glyceraldehyde 3-phosphate Dehydrogenase, Cont’d

35

Mechanism of Glyceraldehyde 3-phosphate Dehydrogenase

36

Mechanism of Glyceraldehyde 3-phosphate Dehydrogenase

Electron sink (Stored 2 electrons and one H+). Source & Where?

HisSHCys C

OH

OHC

CH2OP

H

GAP

B:H

N

NH

HisCys C O

H

OHC

CH2OP

H

S H

Tertahedral Intermediate

N

NH2+

O

NAD+

N

NB:

BH

R

Cys increases the acidity of GAP, but is not involved in the redox reaction

37

P OHO

OH

O

NADH + H

HisCys CO

OHC

CH2OP

H

S

P OH

OH

O

O

BH

NN

H

BH

Acyl-thioester Intermediate

38

HisCys CO

OHC

CH2OP

H

S

P O

OH

O

O

BH

NN

H

BH

3-Phosphoglyceryl-Enz Intermediate1. 3-PG-Enz is a

destabilized acylthioester

2. It undergoes 3-phosphoglyceryl transfer to one of the oxygen of Pi bound at the active site

39

CO

OHC

CH2OP

H

P O

O

O

O

1, 3 bPG

His

NN

Cys SH

BH

High energy compound (Acylphosphate

has G˚ = - 49.3 kj/mol)

40

Mechanism of Energy Coupling

41

Mechanism of Energy Coupling

1. Mechanism of energy coupling is when a high energy compound is being synthesized (endergonic reaction), is must be coupled with to some other (exergonic) reaction by mean of a common intermediate

2. GAPDH catalyzes two different steps; the favorable oxidation and unfavorable phosphorylation reactions are coupled by the thioester intermediate

42

3. The oxidation reaction is favored by the deprotonation of the hemithioacetal by His-176

4. Thioester intermediate allows a favorable process to drive an unfavorable reaction

5. Thioester intermediate preserves much of high energy released in the oxidation reaction

Mechanism of Energy Coupling, Cont’d

43

Free Energy Profiles for GAP Oxidation

• (A) Hypothetical case with no coupling between the two processes, oxidation and phosphorlyation. The 2nd step must have a large activation barrier, making the reaction very slow

• (B) Actual case with the two reactions coupled through a thioester intermediate

Mechanism of Energy Coupling, Cont’d

44

Riboflavin Coenzymes

45

Riboflavin in Foods

46

Riboflavins

• Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derived from riboflavin (Vit B2)

• Flavin coenzymes are involved in oxidation-reduction reactions for many enzymes (flavoenzymes or flavoproteins)

• FAD and FMN catalyze one or two electron transfers

47

Riboflavin and its coenzymes

(a) Riboflavin, (b) FMN (black), FAD (black/blue)

48

Reduction, reoxidation of FMN or FAD

49

Biosynthesis of FAD

Riboflain (Vit B2)

50

N

N

NH

N O

O

OH

OH

HO

OH

1

5

10

4a

5'

N

N

NH

N O

O

O

OH

HO

OH

O-

P

O

P OO

O-O

HO

N

OH

O

N

N

N

NH2

N

N

NH

N O

O

O

OH

HO

OH

O-

P

O

O-

Riboflavin Flavin Mononucleotide (FMN) Flavin Adenine Diphosphate (FAD)

N

N

NH

N

CH3

O

OLumiflavin

NH

HN

NH

N NH2

O

OH

HO H

5,6,7,8-tetrahydrobiopterin

N

NH

N O

O

OR

OH

HO

OH

5-Deazaflavins(more closely related to NAD)

Flavin Coenzyme: Vitamin B2, one- and two-electron transfer

tricyclic flavin ring system: isoalloxazine

51

One and Two Electron Reaction of Flavin

52

Reaction of Flavin with O2

53

1. Oxidases: reduced flavin cofactor re-oxidized directly by O2

2. Dehydrogenase: reduced flavin re-oxidized by another group, i.e.,

3. Mixed Function Oxidase: reduced flavin reacts with O2 to give aflavin-4a-hydroperoxide (Fl-OOH) which oxidizes the substrates by transferring an oxygen atom to the substrate. Overall, O2 is“split” with oxygen atom being incorporated in the oxidized substrate, the other oxygen atom ends up as water

4. Electron-Transfer Flavoproteins (ETF):

O2

R-S-S-R

FlH-red2 R-SH

FloxH2O2

R-S-S-R

Classifications of Flavoenzymes

54

Oxygen:

O O O O3 1

triplet singlet

spin: s = 2n +1spin of an electron = ± 1/2

For oxidases and mixed function oxidases

For mixed function oxidases, the flavin 4a-hydroperoxide is theoxygen-transfer (oxidizing) agent

pKa H2O2 ~ 11.6

O O3

NH

N

NH

NR

O

O

+

electron-tranfer

NH

N

NH

NR

O

O

O O+1

spininversion

NH

N

NH

NR

O

O

OO

H+

N

N

NH

NR

O

O

OHO

Flavin-4a-hydroperoxide

for oxidases

HB:H+

N

N

NH

NR

O

O+ H2O2

55

For mixed function oxidases (monooxygenases), the oxidized flavin Reducing agent is often NAD(P)H and is usually supplieds a separate enzyme

NAD

FAD

N

N

NH

NR

O

O

NH

N

NH

NR

O

O

NAD(P)H NAD(P)

N

NHN

NO

O

R

NicotinamideE°'~ -0.32 V

FlavinE°'~ -0.20 V

N

HH

H2NOC

R

56

• Three main different mechanisms have been proposed for the reaction catalyzed by this flavoenzymes:

• (1) Carbanion formation mechanism: by abstraction of the H+ of the substrate

• (2) Direct hydride-transfer mechanism• (3) Concerted mechanism (1 and 2 together)

Mechanism of Flaovenzymes

57

Carbanion Mechanism

58

D-Amino Acid Oxidase

59

D-Amino Acid Oxidase,Cont’d

• D-Amino acid oxidase (EC 1.4.3.3, DAAO) catalyzes the dehydrogenation of D-isomer of amino acids to give the corresponding -imino acids and, after subsequent hydrolysis, -keto acids and ammonia

• It spreads from yeasts to human and it is not present in bacteria nor in plants

60

• Recently mammalian D-amino acid has been connected to the brain D-Ser metabolism and to the regulation of the glutamatergic neurotransmission

D-Amino Acid Oxidase, Cont’d

61

Structure of D-Amino Acid Oxidase

Human DAAOYeast DAAO

http://en.wikipedia.org/wiki/Image:1c0p_DAAO_dimer.jpg

62

• D-Ala is located above the reduced flavin Re-side

• (- - -) lines denote hydrogen bonds involved in substrate fixation

Structure of D-Amino Acid Oxidase, Cont’d

Yeast DAAO

http://www.pnas.org/content/vol97/issue23/images/large/pq2300387002.jpeg

63

Stabilization of the Negative Charge in the Active Site by Arg-285

Yeast DAAO

DAAO + D-Ala

http://www.jbc.org/content/vol275/issue32/images/large/bc3007236001.jpeg

64

Potential of the DAAO-based Selection system

65

Potential of the DAAO-based Selection System

66

Potential of DAAO-based Selection System, Cont’d

• (a) Growth of nontransgenic wild-type plants lacking DAAO activity is inhibited by D-amino acids such as D-Ala but is not affected by others such as D-Ile

• In contrast, plants expressing the transgenic DAO1 gene detoxify D-Ala and survive (positive selection), whereas they metabolize D-Ile to toxic compounds that kill the plants (negative selection)

67

• (b) Plants that have integrated a gene of interest together with the DAO1 marker are first detected by positive selection

• Subsequent negative selection identifies plants from which the no-longer-desirable selection marker has been removed (e.g., by genetic segregation or site-specific recombination systems), leaving the gene of interest as the only transgenic sequence in place

Potential of DAAO-based Selection System, Cont’d

68

L-Alanine D-Alanine

Structure of D- and L-Alanine

69

Structure of D- and L-Alanine (Cont’d)

D-AlanineL-Alanine

70

D-Amino Acid Oxidase

• D-Amino acids are not normal metabolites in mammalian cells

• D-Amino acids librated from bacterial cells that have been lysed by macrophages

• D-AA oxidase catalyzes the reaction by N-nucleophile mechanism but not by C-nucleophile mechanism because X-ray structure shows no active site base

71

• The standard reduction potential of the flavin in D-amino acid oxidase, a flavoprotein, is about 0.0 V

• Remember, the more positive the standard reduction potential, the more likely the substrate will be reduced and hence act as an oxidizing agent

• FAD in D-AA oxidase is a better oxidizing agent than free FAD

D-Amino Acid Oxidase,Cont’d

72

• The Kd for binding of FAD to the enzyme is 10-7

M compared to the Kd for binding of FADH2, which is 10-14 M

• By gaining electrons, the FAD binds more tightly, which preferentially stabilizes the bound FADH2 compared to the bound FAD

• This shifts the equilibrium of FAD → FADH2 to the right, making the bound FAD a stronger oxidizing agent

D-Amino Acid Oxidase, Cont’d

73

Reaction of D-Amino Acid Oxidase

D-Alanine

C

O

OH CNH3

HCH3

C

O

OH CNH2

CH3

C

O

OH CNH3

HCH3

Enz-FAD Enz-FADH2

Enz-FAD

ReoxStep

74

Reaction of D-AA Oxidase, Cont’d

Hydrogen peroxide

O2

H2O2

+C

O

OH CNH2

CH3

EnzFAD

Enz-FAD

H2O2

75

Reaction of D-AA Oxidase, Cont’d

Nonenzymatic Rnx

Pyruvate

Pyruvate imine

C

O

OH CNH2

CH3

H2O

NH4

C

O

OH CCH3

O

76

Mechanism of D-Amino Acid Oxidase

77

C-nucleophile mechanism. Amino acid enolate adds to the flavin 4a-position. The flavin then acts as a leaving group

N

N

NH

NR

O

OR CO2H

H2N H :B

R CO2H

H2N

H+ NH

N

NH

NR

O

OR CO2HH2N

NH

N

NH

NR

O

OR CO2H

H2NH2O

R CO2H

ONH4++

pKa~ 25

X-ray structure shows NO active site base


78


(Hydride Transfer Mechanism)N-Nucleophile Mechanism

79

N

NNH

NH3C

H3CO

O

R

BH+

Arg-285C

Ser-335

C COO

CH3

H

NH

H

HB:

C COO

CH3

N

H

H

N

NNH

NH3C

H3CO

O

R H

H

O2O O

B:

BH+

OH

NH2H2N

D-Amino Acid

FAD FADH2

Electron sink (Stored 2 electrons and 2 H+). Source & Where?

Pyruvate imine

Redox step

80

Mechanism of D-AA Oxidase, Cont’d

FAD

Hydrogen peroxide

N

NNH

NH3C

H3CO

O

R

H

O OHB:

BH+

H2O2

N

NNH

NH3C

H3CO

O

R

81

Mechanism of D-AA Oxidase, Cont’d

C

O

OH CNH2

CH3

H2O

NH4

C

O

OH CCH3

O

Pyruvate imine

Non-enzymatic Rxn

Pyruvate

82

p-hydroxybenzoate Hydroxylase

83

• p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein involved in degradation of aromatic compounds, and it has become a model for enzymes involved in the oxygenation of a substrate

• It is an important enzyme in the microbial biodegradation of a wide variety of aromatic chemicals, including pollutants and lignin, a major component of wood and so among the most abundant of all biopolymers

p-hydroxybenzoate Hydroxylase

84

• Enzyme structure is unusual because there is no recognizable domain for the binding of NADPH involved in the reaction

• The flavin ring structure moves substantially in the active site, probably to enable substrate and product exchange into this site and possibly to regulate the reduction of the flavin by NADPH

p-hydroxybenzoate Hydroxylase, Cont’d

85

• A chain of H-bonds can connect p-hydroxy-benzoate in the active site of the enzyme with the protein surface

• This chain is responsible for the reversible formation of substrate phenolate anion observed in the active site and partly responsible for the reactivity of this substrate

p-hydroxybenzoate Hydroxylase, Cont’d

86

• An OH group (center) is transferred from a flavin cofactor (right) to the substrate (left)

• The transition state is stabilized by a hydrogen bond interaction with a key group in the enzyme (shown as a dotted line)

Stabilization of Transition State of p-hydroxybenzoate Hydroxylase

87

Reaction of p-hydroxybenzoate Hydroxylase

COOHO

NADPHNADP

O2 H2O COOHO

HOEnz-FADH2 Enz-FAD

+ +

4-hydroxybenzoate 3,4-dihydroxybenzoate

Reductase

Hydroxylase

88

Reaction Steps of p-hydroxybenzoate Hydroxylase

Reduced form

Reduced form

89

Mechanism of p-hydroxybenzoate Hydroxylase

(Monooxygenase)

90

N

NNH

NH3C

H3CO

O

R

N

NNH

NH3C

H3CO

O

R H

H

O2 O O

B:

BH+H

H

FADH2

Mechanism of p-hydroxybenzoate Hydroxylase (Monooxygenase)


91

Hydroxybenzoate

Mechanism of p-HB Hydroxylase (Cont’d)

Electrophilic oxygenN

NNH

NH3C

H3CO

O

R

H O OH

BH+

CCO

O H

N

NNH

NH3C

H3CO

O

R

H O

CCO

OHO

H

H

B:

BH+

B:

BH+

92

NADPH

FAD


N

NNH

NH3C

H3CO

O

R

NADPH

HO

COO

HO

N

R

NH2

OH H

..

BH+

+H2O

Mechanism of p-HB Hydroxylase, Cont’d

93

N

NNH

NH3C

H3CO

O

R

H

H

N

NH2

O

+

R

Reductase

NADP+

FADH2

Mechanism of p-hydroxybenzoate Hydroxylase, Cont’d

94

Glutathione Reductase

95

Biosynthesis of Glutathione

96

Function of Glutathione

• Glutathione (GSH) is an important tripeptide (Glu, Cys, and Gly ) present in significant concentrations in all tissues

• The function of GSH is to protect cells from oxidative stress or the presence of ROS which might otherwise damage them

• The oxidizing agents react with the -SH group of Cys of the GSH instead of doing damage elsewhere

97

• Many foreign chemicals get attached to GSH, which is really acting as a detoxifying agent

• GSH reductase (from human RBC) is dimer of identical 478-residue subunits (52.4KD per monomer) are covalently linked by an intersubunit disulfide bond

• Each subunit contains FAD- and NADPH-binding domains that composed of a babab


98

• The two S atoms of the redox-active residues are in yellow spheres

• The FAD prosthetic groups are in orange color near the active disulfide bridge


http://www.answers.com/topic/glutathione-reductase-png

99

• Each subunit is organized into five domains

• The two subunits are covalently linked by a disufide bridge

• The binding sites for NADPH, GSSG and FAD are indicated


100

Glutathione Reductase Catalytic Cycle

101

Reaction of Glutathione Reductase

GS-SG

102

Mechanism of Glutathione Reductase

(Indirect-NAD-Hydride Transfer)

103

Mechanism of Glutathione Reductase(Indirect-NAD-Hydride Transfer)

His

SS

Cys-58 Cys-63

N

NNH

NH3C

H3CO

O

R

N

R

NH2

OH H

..

3 BH+

N

NH

B:

SS

N

NNH

NH3C

H3CO

O

R

H3

H

NADP+

N

N

H

FAD FADH2

NADPH Electron sink (Stored 2 electrons and 2 H+). Source & Where?

104

Mechanism of Glutathione Reductase, Cont’d

BH+

SS

N

NNH

NH3C

H3CO

O

R H

3H

N

NGSSG

G S GS

105


B:

SS

N

NNH

NH3C

H3CO

O

R H

N

N

H

G S

GS3H

106


BH+

SS

N

NNH

NH3C

H3CO

O

R

N

N

G S

H

GSH

SS

N

NNH

NH3C

H3CO

O

R

N

N

H

107

NAD/NADH versus FAD/FADH2

108

NAD/NADH vs FAD/FADH2

• FAD binds tightly to the enzymes (sometimes covalently attached to Cys or His through C-8a) so as to control the nature of the oxidizing/reducing agent that interact with them

• Because FADH2 is susceptible to reaction with dioxygen (O2)

• FAD/FADH2 can form stable free radicals arising from single electron transfers

109

• O2 in the cell won't react with FAD in the cytoplasm

• If bound FAD is used to oxidize a substrate, the enzyme would be inactive in any further catalytic steps unless the bound FADH2 is reoxidized by another oxidizing agent

NAD/NADH vs FAD/FADH2, Cont’d

110

• FAD has a more positive reduction potential than NAD

• It is used for more demanding oxidation reactions, such as dehydrogenation of a C-C bond to form an alkene (C = C)


111

• FAD can exit as FAD or FADH2 whereas NAD can exit as NAD or NADH

• FAD can carry out 1 electron and 1 proton or 2 electrons and 2 protons whereas NAD can carry out only 2 electrons and 1 proton


112

• The standard reduction potential for flavin enzymes varies from - 465 mV to + 149 mV

• Because the FAD is tightly bound to the enzyme so its tendency to acquire electrons depends on its environment

• Comparing to the reduction potential of free FAD/FADH2, which in aqueous solution is -208 mV


113

• The standard reduction potential of the flavin in D-amino acid oxidase is about 0.0 V

• FAD in D-amino acid oxidase is a better oxidizing agent than free FAD

• NADH does not react well with O2, since single electron transfers to/from NAD/NADH produce free radical species which can not be stabilized effectively whereas FAD reacts with O2


114

Pterin Coenzyme

115

Pterin Coenzyme

• Coenzyme has a 3-carbon side chain at C-6

• Not vitamin-derived, but synthesized by some organisms

(5,6,7,8, Tetrahydrobiopterin, THB or BH4)

116

Pterin, Folate and Tetrahydrofolate (THF or FH4)

117

Biosynthesis of Tetrahydrobiopterin (THB)

118

Biosynthesis of THB, Cont’d

119

Biosynthesis of THB, Cont’d

120

• THB is the coenzyme for Phe 4-hydroxylase (PAH), Tyr 3-hydroxylase, andTrp 5-hydroxylase; the latter two are key enzymes in the biosynthesis of biogenic amines

• THB serves as the cofactor for nitric oxide synthase and glyceryl-ether monooxygenase

• THB can react with O2 to form an active oxygen intermediate that can hydroxylate substrates

Structure of THB

121

Phenylalanine Hydroxylase

122

Structure of Phenylalanine Hydroxylase (PAH)

• It catalyzes the conversion of Phe to Tyr • It is regulated by Phe, THB, and

phosphorylation• Four subunits of PAH interact to form a

tetramer, which is the functional unit for this enzyme

• It is non-heme metallenzyme

123

• PAH includes a non-heme iron atom at its active site

• Fe bound to His N, Glu O and water O

• O2, THB, and the iron atom in the ferrous (Fe2) oxidation state participate in the hydroxylation

• O2 react initially with THB to form a peroxy intermediate

His

His Glu

7,8-dihydrobiopterin

Phenylalanine Hydroxylase PDB 1DMW

Structure of PAH, Cont’d

124


• Tyrosine, an essential nutrient for individuals with PKU, must be supplied in the diet

Transaminase Phenylalanine Phenylpyruvate (Phenylketone) Phenylalanine Deficient in Hydroxylase Phenylketonuria

Tyrosine Melanins Multiple Reactions

Fumarate + Acetoacetate

125

• Each molecule in the tetramer is organized into three domains: – Regulatory domain– Catalytic domain where the enzyme activity

resides – Tetramerization domain that assembles four

chains into the tetramer• At the heart of each catalytic domain is an

iron ion (Fe) that plays an important role in the enzyme action


126


127


128

Reactions of PAH and THF

129

• Genetic deficiency of PAH leads to the disease phenylketonuria (PKU)

• Phe and phenylpyruvate accumulate in blood and urine

• Mental retardation results unless treatment begins immediately after birth

• Treatment consists of limiting phenylalanine intake

Deficiency of PAH

130

• High concentration of Phe:– Can cause neurologic damage – Inhibits Tyr Hydroxylase, on the pathway for

synthesis of the pigment melanin from Tyr– Individuals with PKU have light skin and hair color

• The biosynthesis of the neurotransmitters (dopamine, adrenaline, and noradrenaline) from dietary Phe (into Tyr) is initiated by the PAH

• Tyr becomes an essential nutrient for individuals with PKU

Deficiency of PAH, Cont’d

131

• An unexpected aspect of the PAH reaction is that a 3H atom ends up on C3 rather than being lost to the solvent by replacement for the OH group

• The mechanism is called NIH shift or 1,2 shift mechanism

Experimental Evidence for 1,2-Shift Mechanism

132

D

CO2-

NH3 HO

CO2-

NH3

D

~50% D- incorporation

CO2-

NH3HODB:

CO2-

NH3HOThis mechanism would require NO D in the product

Electrophilic substitution analogous top-hydroxybenzoatehydroxylase

Experimental Evidence for 1,2-Shift Mechanism (Cont’d)

133

D

CO2-

NH3NH

HN N

NH

O

NH2OH

OH

H

OHO

+CO2

-

NH3O

D

H

CO2-

NH3O

D

HH

B

+

1,2-hydrideshift

CO2-

NH3HOD H

CO2-

NH3HOH (D)

~50% D- incorporation

Experimental Evidence for 1,2-Shift Mechanism, Cont’d

134

Mechanism of PAH(Mono-oxygenase)

135

Mechanism of PAH(Mono-oxygenase)

H2N

OH

H

H

CH C H CH3

OHOH

N

N N

N

OO

B:

H2N

O

H

H

N

N N

N

OO

His-290

Glu-330

His-285Fe2+

H2O H2O

H2O

RTHB

Pterin hydroperoxideElectron sink (Stored 1 electron and H+). Source & Where?

136

Mechanism of PAH, Cont’d

H2N

O

H

H

N

N N

N

O

H2O

Fe2+

H2O

O

RH2OO

H

H

N

N N

N

OHR

NH

H B:

BH+

H2O

Fe4+

H2O

O2

+ HOHPterin-4a-carbinolamine

Oxyferry

Electron sink (Stored 1 electron and 1 H+). Source & Where?

137


Fe4+

O

RH3H2O

Fe4+

H2O

O2

Phe

B:

O

H

N

N N

N

NH

H

BH+ R

Oxyferry

Phe

Dehydrogenase

13

2

Oxyferry


138

H2N

OH

H

H

CH C H CH3

OHOH

N

N N

N

H3

NADPH3

NADP+


1

DHB reductase

THB


139

2

DHB reductase

THB

O

H

N

N N

N

NH

R

NADP+

NADPH

HB:

BH+

O

H

N

N N

N

NH

R

H

H

H2N

OH

H

H

CH C H CH3

OHOH

N

N N

N

DHB

Electron sink (Stored 2 electrons and H+). Source & Where?

140

RH3

HO

RH3

H

OH

H

RH3

H

OH

RH3

H

O

R

3

OH

H

TyrH

Carbonion at C3

Hydrogen ion migration

Resonance-stabilized oxonium ion

Epoxide

3

141

Tetrahydrofolate (THF)

142

• Folate is obtained primarily from yeasts and leafy vegetables as well as animal liver

• Animal cannot synthesize PABA nor attach glutamate residues to pteroic acid, thus, requiring folate intake in the diet

• When stored in the liver or ingested folate exists in a polyglutamate form

Biosynthesis and Absorption of THF

143

Folic Acid (Vitamin B9)We cannot synthesize

144


• Vitamin folate is found in green leaves, liver, yeast

• The coenzyme THF is a folate derivative where positions 5,6,7,8 of the pterin ring are reduced

• THF contains 5-6 glutamate residues which facilitate binding of the coenzyme to enzymes

• THF participates in transfers of one carbon units at the oxidation levels of methanol (CH3OH), formaldehyde (HCHO), formic acid (HCOOH)

145


• THF is an important coenzyme in nitrogen metabolism

• Biotin transfers carbon in its most oxidized state-carbon dioxide (CO2)

• SAM can transfer carbon in its most reduced state – methyl groups (but the methyl group comes from 5-methyl-THF)

• THF transfers one-carbon groups in intermediate oxidation states and sometimes as methyl groups

146

• Intestinal mucosal cells remove some of the glutamate residues through the action of the lysosomal enzyme, conjugase

• The removal of glutamate residues makes folate less negatively charged (from the polyglutamic acids) and therefore more capable of passing through the basal lamenal membrane of the epithelial cells of the intestine and into the blood

THF, Cont’d

147

• Folate is reduced within cells (principally the liver where it is stored) to THF through the action of dihydrofolate reductase (DHFR), an NADPH-depending enzyme

THF, Cont’d

148

Formation of THF from folate

149

• One-carbon derivatives of THF

• Note: these are positions 5,6,7,9 and 10

Continued next slide

Different Forms of THF

151

THF, Vitamin B12 and SAM

THF THF-CDHF

dUMPdTMPNADPHNADP+ A

Purine precursors

Purines (C2 and C8)

B

GlySer C

B12

B12-CH3

Homocysteine

Meth

SAMSAH

CH3

NorepinephrineGuanidinoacetateNucleotidesPhosphatidylethanolamineAcetylserotonin

Methyated nucleotidesCreatine

PhosphatidylcholineMelatonin

Epinephrine

D

Glucose

Ser

Gly1

His

Formimino-GluGlu NH4

+

2NH4

+Gly

3CO2 +

+

Epinephrine

Formaldehyde4 Trp

Formate

5

Recipients of carbon

Sources of carbon(1 - 5)

(A - D)

152

THF in the Metabolism of One-Carbon Units

methyl

methylene

formyl

Single carbon groups can be carried on N-5,N-10, or bridged between N-5 and N-10

Carbon units are obtained from a variety of sources BUT most activated single carbon units are obtained from the beta carbon of serine

Once a single carbon unit has been activated by attachment to tetrahydrofolate it can be used directly in a biosynthetic reaction or it can undergo interconversions to different oxidation states

153

Folate Deficiencies

• Early 1990s: Epidemiological studies demonstrated correlations between folate deficiencies and increased risk of myocardial infarctions– heart attacks

• These same individuals also had elevated levels of homocysteine

154

• Homocysteine accumulates in folate deficient individuals because of a decrease in the ability of the methionine synthase reaction to function (due to lack of THF)

• Homocysteine causes heart damage by an unknown mechanism

Folate Deficiencies, Cont’d

155

• Folate deficiencies during embryogenesis cause a significant proportion of neural tube defects and consequent failure of the nervous system to develop properly

• This is most likely due to inability to synthesize adequate amounts of thymine nucleotides

Folate Deficiencies, Cont’d

156

Homocysteine

157

Homocysteine

Homocysteinuria• Rare; deficiency of cystathionine b-synthase• Dislocated optical lenses• Mental retardation• Osteoporosis• Cardiovascular disease death

High blood levels of homocysteine associated withcardiovascular disease

• May be related to dietary folate deficiency• Folate enhances conversion of homocysteine to methionine

158

Enzyme Deficiencies Causing Homocystinuria

159

Thymidylate Synthase

160

Reaction of Thymidylate Synthase

161


• Thymidylate synthase sits at a junction connecting dNTP synthesis with folate metabolism

• It has become a preferred target for inhibitors designed to inhibit DNA synthesis

• An indirect approach is to employ folate precursors or analogs as antimetabolites of dTMP synthesis

• Purine synthesis is affected as well because it is also dependent on THF

162

Thymidylate Synthase, Cont’d

• Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase

• The 5-CH3 group is ultimately derived from the -carbon of serine

163

• 5-Fluorouracil (5-Flu) is a thymine analog• It is converted in vivo to 5'-fluorouridylate by a

PRPP-dependent phosphoribosyltransferase, and passes through the reactions of dNTP synthesis, culminating ultimately as 2'-deoxy-5-fluorouridylate, a potent inhibitor of dTMP synthase

• 5-Flu is used as a chemotherapeutic agent in the treatment of cancer


164

• Similarly, 5-fluorocytosine is used as an antifungal drug because fungi, unlike mammals, can convert it to 2'-deoxy-5-fluorouridylate

• Further, malarial parasites can use exogenous orotate to make pyrimidines for nucleic acid synthesis whereas mammals cannot

• 5-fluoroorotate is an effective antimalarial drug because it is selectively toxic to these parasites


165


• Precursors and analogs of folate employed as antimetabolites:– Sulfonamides– Methotrexate– Aminopterin– Trimethoprim

• They bind to DHF reductase with about one thousand-fold greater affinity than DHF and thus act as virtually irreversible inhibitors

168

• Sulfa drugs, or sulfonamides, owe their antibiotic properties to their similarity to p-aminobenzoate (PABA), an important precursor in folate synthesis


• Sulfonamides block folate formation by competing with PABA

169

Human thymidylate synthase

http://www.medlibrary.org/medwiki/Image%3AThymidylate_synthase_1HVY.png

170

Mechanism of Thymidylate Synthase

171

Mechanism of Thymidylate Synthase

N5, N10-Methylene-THF

dUMP


Cys

N

N

N

N

H2N

O

H

HCH2

N RH2C

R..BH+

S

N

N

N

N

H2N

O

H

HCH2

N R

R

H2C

H

O

HH

OH H

HH

N

NH

O

O

OCH2P

O

OHO

H

Cys

S

HB:

172

Mechanism of Thymidylate Synthase, Cont’d


N

N

N

N

H2N

O

H

HCH2

N R

R

H2CH

O

HH

OH H

HH

N

NH

O

O

OCH2PO

OHO

Cys

S

N

N

N

N

H2N

O

H

H CH2

NH2C

H

OCH2PO

OHO

Cys

S

BH+

H

B:

O

HH

OH H

HH

N

NH

O

O

R

R

173

Mechanism of Thymidylate Synthase, Cont’d

N

N

N

N

H2N

O

H

HCH2

H2C

OCH2PO

OHO

Cys

S

R

RN

H

H

O

HH

OH H

HH

N

NH

O

O

B:H

BH+

Cys

S

H

N

N

N

N

H2N

O

H

HCH2 R

RN

H

+OCH2P

O

OHO O

HH

OH H

HH

N

NH

O

OH3C

dTMP


7, 8-DHF

Hydrogen transfer

174

Mechanism of Thymidylate Synthase Inhibition by 5-

Fluoro-dUMP

175

Mechanism of Thymidylate Synthase Inhibition by 5-Fluoro-dUMP

N5, N10-Methylene-THF

FdUMP

Cys

N

N

N

N

H2N

O

H

HCH2

N RH2C

R..BH+

S

N

N

N

N

H2N

O

H

HCH2

N R

R

H2C

H

O

HH

OH H

HH

N

NH

O

O

OCH2P

O

OHO

H

Cys

S

HB:

F

176

N

N

N

N

H2N

O

H

HCH2

N R

R

H2C

H

O

HH

OH H

HH

N

NH

O

O

OCH2PO

OHO

Cys

S

N

N

N

N

H2N

O

H

H CH2

NH2C

H

OCH2PO

OHO

Cys

S

B:

O

HH

OH H

HH

N

NH

O

O

R

R

F F

X

Mechanism of Thymidylate Synthase Inhibition by 5-Fluoro-

dUMP

Inhibition by Flu-dUMP results from the electronegativity of the fluorine which generates a C-F bond at C5 that cannot be broken

177

• Most normal mammalian cells, requires less dTMP and so are less sensitive to inhibitors that inhibit thymidylate synthase or DHF reductase with exceptions are:– Bone morrow cells that constitute the blood-

forming tissue and much of the immune system– Intestinal mucosa– Hair follicles


dUMP

178

• 5-FludUMP is irreversible inhibitor of thymidylate synthase

• It binds to the enzyme and undergoes the 1st two steps of the normal enzymatic reaction

• In step 3, the enzyme cannot abstract the F atom as F+ because F is the most electronegative element, so that enzyme is bound covalently with F and forming Enz-FludUMP-THF ternary complex


dUMP

179

Enz-FludUMP-THF Ternary Complex

• The slide shows the X-ray structure of this Enz-FludUMP-THF ternary complex :– Its active site region is

shown with helices (yellow), -stands (organe), and other polypeptides (blue)

– C-5 and C-6 of FludUMP (green spheres) form covalent bond (red) with CH2 group (blue) and S of Cys (yellow spheres)

180

Orientation of Substrate and Coenzyme in the Active Site of


181

Regeneration of N5, N10-Methylenetetrahydrofolate

182

Relationship between THF, Vit B12 and SAM

183

Serine Hydroxymethyltransferase

184

Serine Hydroxymethyltransferase (SHMT)

• It catalyzes the reversible conversion of of Ser to Gly using PLP and THF as coenzymes

• It is unusual enzyme because it utilizes PLP for C-C bond formation at the oxidation level of formaldehyde

• It is largely responsible for the provision of cellular one-carbon methylene

• Because in the reverse reaction it can be used to generate N5-methylene-THF from Ser

185

• It is a part of the -class of PLP enzymes

• In the mechanism, Ser is bound to PLP, and is converted to Gly by cleaving off a formaldehyde from the Ser side chain, with the formaldehyde binding to THG, converting it to N5,N10-methylene THF

SHMT, Cont’d

186

SHMT, Cont’d

187

• SHMT is a common enzyme complex, with homologous structures present in both prokaryotes and eukaryotes, including humans

• These enzymes, though genetically dissimilar, have matching secondary and tertiary structures between the subunits of different species

SHMT, Cont’d

188

• The final enzyme complex of the prokaryotes and eukaryotes also differs, with the prokaryotes tending to form tight dimers of four subunits, while the eukaryotes form tetramers

• In eukaryotes, SHMT is present in both the cytosol and the mitochondria

SHMT, Cont’d

189

• The 2 isoenzymes of SHMT exist in cytosolic and mitochondrial compartments (cSHMT and mSHMT, respectively) and may have arisen from a gene duplication event after the divergence of bacterial and eukaryotic proteins

• Communication between the cytosolic and mitochondrial compartments in one-carbon metabolism is achieved using metabolites that can cross the mitochondrial membrane, primarily Ser, Gly, and formate

SHMT, Cont’d

190

• Serine is considered the major source of one-carbon units, which are generated through the production of glycine and 5,10-methyleneTHF by both the cytosolic and the mitochondrial forms of SHMT, but primarily by mSHMT

• In eukaryotic systems, cSHMT generally operates in the direction of Ser synthesis, whereas mSHMT primarily works in the opposite direction

SHMT, Cont’d

191

Serine Hydroxymethyltransferase

One subunit of the dimeric enzyme

192

SHMT, Cont’d

193

Mechanism of Serine Hydroxymethyltransferase

194

Mechanism of Serine Hydroxymethyltransferase

N

OH

CH3

H

C

OP

O

OO

LysNH

H

CN COO

CH2

HH

H

OHB:N

OH

CH3

H

C

OP

O

OO

LysNH2

H

C COO

H

H2CHO

N

B:..


PLP-Enz Schiff base

(Aldimine)Ser PLP-Ser Schiff base

195

Mechanism of Serine Hydroxymethyltransferase, Cont’d

N

OH

CH3

H

C

OP

O

OO

LysNH2

H

C COOH2CHO

N

BH+

..

..

N

OH

CH3

H

C

OP

O

OO

Lys

NH2

H

C COO

N

..

C

H H

THF H2OR

N

N

N

N

H2N

OCH2NH

H

H

H

THF

B:

Quinonoid


Ketimine

196


N

OH

CH3

H

C

OP

O

OO

LysNH2

H

C COO

N

..

C

RN

N

N

N

H2N

OCH2NH

H

..

H2 BH+

N

OH

CH3

C

OP

O

O

NH2

H

C COO

N

..

C

N

N

N

N

H2N

OCH2NH

H

H2

Lys

H

HH

O

R

B:H H

C

R

N

N

N

N

H2N

O

H2

H

HC

H2

N

PLP-Ser-Schiff base


5N, 10N-methylene-THF

197


Quinonoid


PLP-Gly-Schiff base

N

OH

CH3

H

C

OP

O

O

LysNH2

H

C COO

N

..

..

H

BH+

H2O

N

OH

CH3

H

C

OP

O

OO

Lys

NH2

H

C COO

N

..

H

H

OH

B:

BH+

H

198


Gly

PLP-Enz Schiff base

H2

C COO

N

H

H N

OH

CH3

H

C

OP

O

OO

LysNH2

H

..O

N

OH

CH3

H

C

OP

O

OO

LysNH

H

1 part 2 coenzymes-dependent enzyme mechanism professor a. s. alhomida disclaimer the texts, tables...

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