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

1

Part 1Coenzyme-Dependent Enzyme

MechanismProfessor A. S. Alhomida

Part 1Coenzyme-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

2

Syllabus

• Instructor: Professor A. S. Alhomida– Office: 2A 62; Tel: 467-5938– E-mail: [email protected]– Web page: faculty.ksu.edu.sa/alhomida

• Textbook: 1. Enzyme kinetics and mechanism. Cook and Cleland, 2007 2. Enzymatic Reaction Mechanisms. Walsh, 19793. Introduction to Enzyme and Coenzyme Chemistry, 2nd Edition.

Bugg, 20044. Contemporary Enzyme Kinetics and Mechanism. Purich, 19835. Structure and Mechanism in Protein Science: A guide to Enzyme

Catalysis and Protein folding. Fersht, 19996. Biochemistry 2nd edition. Garrett and Grisham, Chapter, 14-16

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7. Lechninger's Principles of Biochemistry 4th edition. D. L. Nelson and M.M. Cox., Chapter 6

8. Biochemistry 3rd Edition. Mathews, Holde and Ahern. Chapter: 11

9. Fundamentals of Biochemistry 2nd Edition by Voet and Voet. Chapters: 11, 12

10. Biochemistry 3rd Edition. Zubay. Chapters: 8-1111. Stryer’s Biochemistry, 5th Edition. Berg, Tymoczko and

Stryer. Chaper 8-10.12. Principles of Biochemistry, 4th Edition. Horton, Scrmgeour,

Perry and Rawn. Chapter 5-7

Syllabus, Cont’d

4

• It will focus on article dealing with enzyme mechanisms and you will give a short oral presentation to the class on your analysis of the article

• This project is designed to give you some experience with reading and interpreting original research reports that deal with the study of enzymatic reaction mechanisms and with making an oral presentation of a scientific study using PowerPoint

Oral Presentation Project

5

• Choose an article from a current issue of a biochemical journal

• The article must be an original research report (not a review article) that deals with the study of the mechanism of action of some enzyme and utilizes the techniques of site-directed mutagenesis or site-directed inactivation (transition state analogs)

Oral Presentation Project, Cont’d

6

• Prepare and give a 10-15 minutes oral presentation that gives an overview of the study described in your paper and an explanation of the data that supports the author’s conclusions

• The presentations will be given in class on December 22nd

• Submit a soft copy of your presentation saved in CD disk using PowerPoint


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• Completion Schedule – On Saturday, December 22nd, select a date for

your presentation– Before Saturday, January 5th, submit a photo

copy of your chosen article. I will make copies of articles and distribute them to other members of the class on Saturday, January 12th

• The project will account for 20 out of 70 of your course grade


8

9

Vitamins and Coenzymes

10

11

12

13

14

15

Glycolysis

TCAcycle

Glycogenolysis

aKGDHvit B1,B2,B3

PP avit B6

Glc

GlycogenG1P

R5PTK

vit B1

PDHvit B1,B2,B3

aKGSCoA

Acetyl-CoA

G6P

Pyr

G3PALT

vit B6

Ala

ASTvit B6

OAAsp

vit B6 Glu

PPP

Vitamins in Metabolic Pathways

16

Coenzymes and Vitamins

Apoenzyme + Cofactor Holoenzyme

(protein only) (active)

(inactive)

• Some enzymes require cofactors for activity

(1) Essential ions (mostly metal ions)

(2) Coenzymes (organic compounds)

17

Coenzymes

• Coenzymes act as group-transfer reagents

• Hydrogen, electrons, or other groups can be transferred

• Larger mobile metabolic groups can be attached at the reactive center of the coenzyme

• Coenzyme reactions can be organized by their types of substrates and mechanisms

18

Types of cofactors

19

Inorganic Cations

• Enzymes requiring metal ions for full activity:

(1) Metal-activated enzymes have an absolute requirement or are stimulated by metal ions (examples: K+, Ca2+, Mg2+)

(2) Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples:

iron, zinc, copper, cobalt )

20

Carbonic Anhydrase

21

• Carbon dioxide (CO2) is a major end product of aerobic metabolism

• In mammals, this CO2 is released into the blood and transported to the lungs for exhalation

• While in the blood, CO2 reacts with water• The product of this reaction is a moderately

strong acid, carbonic anhydride (pKa = 3.5), which becomes bicarbonate ion on the loss of a H+

Carbonic Anhydrase

22

• Almost all organisms contain enzyme, carbonic ahydrase, that catalyzes the below reaction

• Cabonic anhydrase accelerates CO2 hydration dramatically at rate as high as kcat =

106 s-1

Carbonic Anhydrase, Cont’d

23

Types of Carbonic Anhydrases

• -Carbonic anydrase:– Found in human, animals, some bacteria and algae– Trimer

• -Carbonic anydrase– Higher plants and many bacteria and E. coli– Has only one conserved His whereas in a has three

His• -Carbonic anhydrase

– Found in bacteria Methanoscarcina thermophila– Has three zn sites similar to carbonic anhydrase

24

Structure of -Carbonic Anydrase

• Zn2+ is coordinated by the imidazole rings of three His residues, His-94, His-96 and His-119

• The primary function of the enzyme in animals is to interconvert CO2 and bicarbonate to maintain acid-base balance in blood and other tissues and to help transport CO2 out of tissues

25

Structure of β-Carbonic Anhydrase

• Found in plans which is an evolutionarily distinct enzyme but participates in the same reaction and also uses a Zn2+ in its active site

• It helps raise the concentration of CO2 within the chloroplast to increase the carboxylation rate of the enzyme Rubisco

• It integrates CO2 into organic carbon sugars during photosynthesis, and can only use the CO2 form of carbon, not carbonic acid nor bicarbonate

26

Structure of -Carbonic Anydrase

Zn bound to three HisThree subunits

http://upload.wikimedia.org/wikipedia/commons/7/7f/Carbonic_anhydrase_1CA2_active_site.png

27

Structure of -Carbonic Anhydrase

• (Left) the Zn site, (Middle) the trimeric structure (A, B, and C) and (Right) the enzyme is rotated to show a top-down view position of the Zn sites

28

Human carbonic anhydrase

29

• How does this Zn2+ complex facilitates CO2 hydration?

• A major clue comes from the pH profile of the enzymatic ally catalyzed CO2 hydration:


30


• At pH 8, the reaction proceeds near its maximal rate

• As the pH decreases, the rate of the reaction drops

• The midpoint of this transition is near pH 7, suggesting that a group with pKa = 7 plays an important role in the activity of this enzyme

31


• The deprotonated (high pH) form of this group participates more effectively in the catalysis

• Although His have pKa value near 7, a variety of evidence suggest that the group responsible for this transition is not His but it is the Zn2+-bound water molecule

• The binding of water to the positively charged Zn2+ center reduces the pKa of the water from 15.7 to 7

32


33

• The lowered pKa generates Zn2+-OH-

complex that is sufficiently nucleophilic to attack CO2 more readily than water does


34

Mechanism of Carbonic Anhydrase

35

Mechanism of Carbonic Anhydrase

Zn2+

His

HisHis

OH

Zn2+

His

HisHis

OH

B:

C OO

B:

CO2

36

• Zn2+ ion promotes the ionization of bound H2O. Resulting nucleophilic OH- attacks carbon of CO2

• The pKa of water drops from 15.7-7 when it is coordinate to Zn2+

• HO- is 4 orders of magnitude more nucleophlic than is water

Mechanism of Carbonic Anhydrase, Cont’d

37

Zn2+

His

HisHis

OH

C

OO

B:

H2O

Zn2+

His

HisHis

OH

C

OO

O

HH

B :

38

Bicarbonate

Zn2+

His

HisHis

OH

C

OO

O

H

BH

O

C OHO

Zn2+

His

HisHis

OH

BH

Tetrahedral intermediate

39

1. The important of Zn2+-OH- comlpex suggests a simple mechanism of CO2 hydration:

2. Zn2+ facilitates the release of a H+ from water, which generates a OH-

3. The CO2 binds to the enzyme’s active site and is positioned to react with the OH-


40

3. The OH- attacks nucleophilically CO2, converting it into bicarbonate ion

4. The catalytic site is regenerated with release of the bicarbonate ion and the binding of another molecule of water


41

Iron in Metalloenzymes• Iron undergoes reversible oxidation and

reduction:Fe3+ + e- (reduced substrate) Fe2+ + (oxidized substrate)

• Enzyme heme groups and cytochromes contain iron

42

Iron in Metalloenzymes, Cont’d

• Nonheme iron exists in iron-sulfur clusters (iron is bound by sulfide ions and S- groups from cysteines)

• Iron-sulfur clusters can accept only one e- in a reaction

43

Iron-sulfur clusters

• Iron atoms are complexed with an equal number of sulfide ions (S2-) and with thiolate groups of Cys side chains

44

Coenzyme Classification

• There are two classes of coenzymes

(1) Cosubstrates are altered during the reaction and regenerated by another enzyme

(2) Prosthetic groups remain bound to the enzyme during the reaction, and may be

covalently or tightly bound to enzyme

45

Classification of Coenzymes in Mammals

(1) Metabolite coenzymes- synthesized from common metabolites

(2) Vitamin-derived coenzymes- derivatives of vitamins (vitamins cannot be synthesized by mammals, but must be obtained as nutrients)

46

Metabolite Coenzymes

47

Metabolite Coenzymes

• Nucleoside triphosphates are examples

ATP 5`-C

48

Reactions of ATP

• ATP is a versatile reactant that can donate its:

(1) Phosphoryl group (-phosphate)

(2) Pyrophosphoryl group (, phosphates)

(3) Adenylyl group (AMP)

(4) Adenosyl group

49

• Nucleotide-sugar coenzymes are involved in carbohydrate metabolism

• UDP-Glucose is a sugar coenzyme. It is formed from UTP and glucose 1-phosphate

(UDP-glucose product next slide)

50

51

Carbon-Carbon Bond Formation

52

Alkylation Reactions

• Methylation is an important transformation in the biosynthesis of many secondary metabolites

• Organic chemists use methyl iodide or methyl sulphonates for methylations

• The biological equivalent is S-adenosyl methionine (SAM)

• The driving force for methyl group transfer is the conversion of a sulphonium ion into a neutral sulphide

53

Alkylation Reactions, Cont’d

54

Aldol and Claisen Reactions

• Reactions between enolates (and their equivalents) with aldehydes or ketones are referred to as aldol reactions

• Reaction of enolates with esters are referred to as Claisen reactions

• They are the most common method to form carbon-carbon bonds

• The biological equivalent of enolates are enamines and coenzyme A

• These are co-factors of aldolase enzymes

55

Enamines

• The side chain of the amino acid lysine carries an amino group

• Reaction with carbonyl compounds leads to imines which tautomerise to give enamines

• Enamines are enolate equivalents and react with carbonyl compounds through nucleophilic attack via their -carbon

• They are used in a very similar way in organic chemistry as shown below for the reaction of a secondary amine (pyrrolidin) with a ketone

56

Enamines, Cont’d

57

Aldol Reactions

• Aldol Reactions Require Several Levels of Control:

• Enol versus carbonyl component: • carbonyl compounds with acidic -protons

can either be deprotonated and react as nucleophiles, or react as electrophiles through their carbonyl group

• If this is not carefully controlled an intractable mixture of products (cross-aldol products) is obtained

58

• Formation of an enamine avoids this problem• The enamine is only nucleophilic• Regioselectivity: enamines are ambident

nucleophiles• They can in principle react through the

carbon or the nitrogen atom• For aldol-type processes, only reactions

through the carbon atom lead to the desired product

Aldol Reactions, Cont’d

59

• In biological systems the regioselectivity is controled by the steric environment of the enzyme active site


60

• Stereoselectivity: The stereochemistry of aldol reactions is highly complex-syn, anti, matched case, mismatched case-are just a few keywords highlighting how difficult it is to control the relative and absolute stereochemistry of aldol products

• In biological systems this is again taken care of by the stereochemistry of the active site of an enzyme


61

S-Adenosylmethionine (SAM)

62

• ATP is a source of other metabolite coenzymes such as S-adenosylmethionine (SAM)

• SAM donates methyl groups in many biosynthesis reactions

Methionine + ATP SAM + Pi + PPi

SAM Biosynthesis

63

Structure of SAM

• Activated methyl group in red

64

Functions of SAM

1. SAM donates the methyl group for many methylation reactions: Methylation of norepinephrins

65

Functions of SAM, Cont’d

2. SAM involves in redox radical-dependent enzymes: Pyruvate formate lyase; Anerobic ribonucleotide reductase

3. Until this point, the only know role for SAM was for methyl group transfer, thus it was surprising to find SAM involved in redox biochemistry

66

Functions of SAM, Cont’d

4. The involvement of SAM in redical biochemistry was first established for Lys 2,3-aminomustase (from C. subterminale) which converts Lys with -Lys

5. Lys 2,3-aminomustase catalyzes the reaction by 1,2 rearrangement mechanism similar to Vit B12-dependent mutase, but didn’t use Vit B12

instead required PLP and SAM for activity and a reduced [4Fe4S] cluster

67

SAM as Methyl Group Donor

– Methylation of bases in tRNA– Methylation of cytosine residues in DNA– Methylation of norepinephrine

68

SAM Cycle

1. SAM synthase (Met adenosyl transferase)

2. Methyltransferase3. S-adenosyl

homocysteinase 4. Homocysteine

methyltransferase

69

Mechanism of SAM Synthase

(Met Adenosyl Transferase)

70


C COO

H

S

O

N

NN

N

OH OH

HHH H

NH2

OO O

O OP

O

OP

O

OP

O

CH2CH2 2

CH3

H3N

MethionineATP

Unusual displacement of triphosphate reaction

:

Nucleophilic attack (SN2 mechanism)

71

• Met is not a sufficient reactive to be a good methyl donor because of the homosysteine mercaptide anion is a poor leaving group

• SAM synthase catalyzes an unusual displacement reaction because of Met sulfur atom attacks nucleophilically on the 5` carbon of ATP to produced the sulfonium compound and and inorganic triphosphate (PPPi ) is formed

Mechanism of SAM Synthase, Cont’d

72

SAM synthase

SAM

PPiPPPiPi


Very good leaving group because of positively charged of S atom

C COO

H

S

CH2 2

CH3

H3N

O

N

NN

N

OH OH

HHH H

NH2

CH2

OO O

O P

O

OP

O

OP

O

O

O O

O P

O

OP

O

O

O

O OP

O

2

O

O OP

O

H2O

Supernucleophile

73

• PPPi is then hydrolyzed by the same enzyme into PPi and Pi making the reaction thermodynamically more favorable

• This is one of two reactions in which a displacement of this kind is known to occur in biological system


74

• The other being the formation of adenosylcobalamin

• The hydrolysis of PPPi drives the reaction to right highly exergoic in the synthetic direction


75

SAM-Dependent Methyltransferase

76

SAM-Dependent Methyltransferase

• The functional roles of methylation are wide ranging and include biosynthesis, metabolism, detoxification, signal transduction, protein sorting and repairing, nucleic acid processing, gene silencing and imprinting

• The majority of methylation reactions are carried out by the SAM-dependent methyltransferases

77

• Human thiopurine S-methyltransferase (TPMT) in complex with SAH

• TPMT is a cytosolic drug-metabolizing enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, azathioprine, 6-thioguanine

SAM-Dependent Methyltransferase, Cont’d

78

• All methylation reactions requiring SAM are simple SN2 (Substitution of nucleophilic bimolecular) displacements

• SAH is a potent inhibitor of all reactions in which a methyl group is transferred from SAM to an acceptor

• It is important to prevent the accummulation of SAH in cells


79

• This is accomplished through the action of S-adenosylhomocysteinase that converts SAH into adenosine and homocysteine

• Homocysteine is converted into Met and adenosine (Ado) into inosine (via SAM cycle)


80

Mechanism of SAM-Dependent Methyltransferase

81

Norepinephrine

SAM

Mechanism of SAM-Dependent Methyltransferase

HO

HO

CH2CH2NH2

OH

..

C COO

H

S

CH2 2

CH3

H3N

O

N

NN

N

OH OH

HHH H

NH2

CH2

Nucleophilic attack (SN2 Mechanism)

82

Mechanism of SAM-Dependent Methyltransferase, Cont’d

HO

HO

CH2CH2NH2

OH

C COO

H

CH2 2

CH3H3N

O

N

NN

N

OH OH

HHH H

NH2

CH2S

+

Epinephrine

S-adenosylhomocysteine (SAH)

83

SAM-Dependent Radical Enzymes

84


• Organic radicals are used by a number of enzymes to catalyze biochemical transformations with high-energy barriers that would be difficult to accomplish through non-radical heterolytic chemistry

• Well known examples include:– Reduction of an alcohol to an alkane catalyzed by

ribonucleotide reductase– Carbon chain rearrangements catalyzed by

methylmalonyl CoA mutase or glutamate mutase

85


• Organic radicals can be generated in enzymes through only three general mechanisms: – Metal-activated oxygen biochemistry– Adenosylcobalamin (Vit B12) biochemistry,

or – Reduction of the sulfonium of SAM

86

Pyruvate Formate Lyase(Formate C-Acetyltransferase)

87

Pyruvate Formate Lyase

• It is an important enzyme (found in Escherichia coli and other organisms) that helps regulate anaerobic glucose metabolism

• Using radical biochemistry, it catalyzes the reversible conversion of pyruvate and CoA into formate and acetyl-CoA

88

Structure of Pyruvate Formate Lyase

• It is a homodimer made of 85 kD, 759-residue subunits

• It has a 10-stranded / barrel motif into which is inserted a finger that contains major catalytic residues

• The active site of the enzyme, elucidated by X-ray crystallography, holds three essential amino acids that perform catalysis:– Gly-734– Cys-418– Cys-419

89

Structure of Pyruvate Formate Lyase, Cont’d

• It is a homodimeric protein (2 x 85 kD) and catalytically inactive when isolated

• Activated enzyme contains one protein radical per dimer at Gly-734 and has a half of the sites reactivity

90

Structure of Pyruvate Formate Lyase, Cont’d

• Three major residues that hold the substrate pyruvate close by Arg-435, Arg-176, and Ala-272), and two flanking hydrophobic residuesTrp-333 and Phe-432

• The active site of enzyme is a similar to that of class I and class III ribonucleotide reductase

91

• The interaction of SAM with the [4Fe–4S]1+ of activated en\yme

• -N and -carboxyl O of Met anchors the SAM to the cluster with the sulfonium interacting with a sulfide from the cluster

SAM-[4Fe4S] ClusterSAM

[4Fe4S] cluster

92

(AE) Activase (DE) Deactivase

Regulationn of Pyruvate Formate Lyase

Radical Radical Gly-734

http://en.wikipedia.org/wiki/Image:PFL_Regulation_Network.jpg

93

Reaction of Pyruvate Formate Lyase

N

H

N

O

HHH

734

[4Fe4S]+

SAM

N

H

N

O

H

734

Gly-734 Gly-734 radical

Pyruvate formate lyase

red

94

Reaction of Pyruvate Formate Lyase, Cont’d

Gly-734 radical

H3C

O

O

OCoA

H3C S

O

CoA

H

O

O

N

H

N

O734

H

Pyruvate

Formate

Acetyl-CoA

Pyruvate formate lyase

95

Mechanism of Pyruvate Formate Lyase

96

Role of Catalytic Residues

Gly-734 (glycyl radical)– Transfers the radical on and off Cys-418, via Cys-

419

• Cys-418 (thiyl radical)– Does acylation reaction on the carbon atom of the

pyruvate carbonyl

• Cys-419 (thiyl radical) – Performs hydrogen-atom transfers

97

Generation of 5`-deoxyadenosyl Radical from SAM by [4Fe4S] Cluster

S Fe

SFe

Fe S

FeS

O

OH

O O

OH

Ad

SH3CHN

H2

S Fe

SFe

Fe S

FeS

O

OH

O O

OH

Ad

SH3CHN

H2

H2C

2

SAM

5`-deoxyadenosyl radical

Enz Enz

98


99


Gly-734

Cys-419

Cys-418

SH

SHRadical transfer from Gly-734 to Cys-419

100

Gly-734

Cys-419

Cys-418 S

SH

HH

Pyruvate

Gly-734

Cys-419

Cys-418

S

SH

H3C

O

O

O

HH

Radical transfer from Cys-419 to Cys-418

Mechanism of Pyruvate Formate Lyase, Cont’d

101

Gly-734

Cys-419

Cys-418

S

HH

H3C

O

O

O

SH

Gly-734

Cys-419

Cys-418

S

H3C O

HH

O

O

SH

Tetrahedral radical intermediate

formate radical intermediate

Thioester (acyl-enzyme)


102

Formate

Radical transfer from Cys-419 to CoA

Gly-734

Cys-419

Cys-418

S

H3C O

HH S

CoA-SO

O

H H

CoA-S H


103

Gly-734

Cys-419

Cys-418

S

H3C O

HH

CoA-S

SH

Gly-734

Cys-419

Cys-418

S

H3C O

HH

CoA-S

SH

Radical transfer from CoA to acetate

Tetrahedral radical intermediate


104

Radical Cys-418

Acetyl-CoA


Gly-734

Cys-419

Cys-418

S

HH SH

C

O

CoA-SH3

105


Gly-734

Cys-419

Cys-418

S

HH SH

e

Gly-734

Cys-419

Cys-418

HH SH

SH

Cys-418 radical enzyme Cys-418 radical inactivated enzyme

106

1. The proposed mechanism begins with radical transfer from Gly-734 to Cys-418, via Cys-419

2. The Cys-418 thiyl radical adds covalently to C-2 of pyruvate, generating an acetyl-enzyme intermediate (which now contains the radical)

3. The acetyl-enzyme intermediate releases a formyl radical that undergoes hydrogen-atom transfer with Cys-419


107

4. CoA comes in and undergoes hydrogen-atom transfer with the Cys-419 radical to generate a CoA radical

5. The CoA radical then picks up the acetyl group from Cys-418 to generate acetyl-CoA, leaving behind a Cys-418 radical

6. Enzyme can then undergo radical transfer to put the radical back onto Gly-734

7. Note that each step is reversible


108

Mechanism for Generating Radical Gly-734

Trasfer radical to inactivated Gly-724 enzyme

From favorodoxin

109

1. Activated enzyme has a novel radical mechanism that utilizes an Fe–S cluster and SAM to facilitate generation of a putative adenosyl radical

2. The Fe–S cluster has a unique iron site in the [4Fe–4S] cluster which is used to coordinate an amino -nitrogen and -carboxyl oxygen to anchor SAM in the active site

Mechanism for Generating Radical Gly-734

110

3. Inner-sphere electron transfer from a bridging sulfide of the [4Fe–4S]1+ cluster to the sulfonium of SAM (AdoMet) causes C–S bond homolysis, which produces a 5′-deoxyadenosyl radical and Met

4. This anchoring allows for the potential inner-sphere electron transfer from the bridging sulfide to the sulfonium of SAM, and facilitates homolytic bond cleavage and creation of the adenosyl radical

Mechanism for Generating Radical Gly-734, Cont’d

111

5. The adenosyl radical abstracts a hydrogen from Gly-734 of enzyme and 5′-deoxyadenosine and Met are replaced with another SAM

6. The active cluster of enzyme has to be in reduced form ([4Fe–4S]1+), which is oxidized to [4Fe–4S]2+ during turnover catalysis

7. The source of the electron is proposed to be a reduced flavodoxin

Mechanism for Generating Radical Gly-734, Cont’d

112

Vitamin-Derived Coenzymes

113

Vitamin-Derived Coenzymes

• Vitamins are required for coenzyme synthesis and must be obtained from nutrients

• Animals rely on plants and microorganisms for vitamin sources (meat supplies vitamins also)

• Most vitamins must be enzymatically transformed to the coenzyme

114

Vitamin C

115

Vitamin C: a Vitamin but not a Coenzyme

• A reducing reagent for hydroxylation of collagen

• Deficiency leads to the disease scurvy

• Most animals (not primates) can synthesize Vit C

116

Vitamin C (ascorbic acid) in Foods

117

Nicotinamide Adenine Dinucleotide

118

Niacin in Foods

119

Niacin in Foods

120

Reduction Reactions

• The biological equivalent of hydride transfer reagents, such as NaBH4, is nicotinamide adenine dinucleotide (NADH) and its phosphorylated analog NADPH

• These are coenzymes of reductase enzymes• The stick model of NAD is taken from an

actual X-ray crystallographic analysis of human alcohol dehydrogenase enzyme

121

Reduction Reactions, Cont’d

122

• The pyridinium ring acts as hydride acceptor in the oxidation step, whilst 1,4-dihydropyridine system acts as hydride donor in the reduction step:


123

• The stereoselectivity of the reduction step relies on the "chiral environment" provided by the active side of the enzyme

• NADH is a coenzyme which is held in the acitve site of the enzyme (alcohol dehydrogenase in this case) by non-covalent interactions

• The image below shows NADH and amino acids in a distance of 5 Å from NADH


124


125

• The image on the left is a close-up view of the residues neighbouring NADH in the active site

• The image on the right shows the whole enzyme (the enzyme is actually a dimer and only one half is shown for clarity)


126

• NAD-dependant Enzymes• Oxidation is the reverse of reduction and the

oxidized form of NADH can act as an oxidant• In oxidation-mode NAD/NADH-dependant

enzymes are referred to as oxidase enzymes• This form is called NAD

Oxidation Reactions

127

• In fact, NAD and NADH have to be reversible redox pairs to allow the coenzyme and the enzyme to act as true catalysts

Oxidation Reactions, Cont’d

128

Cytochrome-P450-dependant Enzymes

• The redox-active species in this class of enzymes is the Fe(III)-Fe(II) couple

• The iron centre is coordinated to a porphorine system

• Together they form the hem coenzyme of oxygenase enzymes (note the difference to oxidase enzymes which contain NAD as coenzyme)

• The name cytochrome P450 is due to the strong absorption at 450 nm of enzymes that contain a hem coenzyme when co-ordinated to carbon monoxide

129

Cytochrome-P450-dependant Enzymes, Cont’d

130

Non-Hem -Ketoglutarate-Dependant Oxygenases

• Enzymes belonging to this class contain an iron centre, but no hem coenzyme

• Isopenicillin-N-synthase, the crucial enzyme in the biosynthesis of penicillin belongs to this class

131

NAD+ and NADP+

• Nicotinic acid (niacin) is precursor of NAD and NADP

• Lack of niacin causes the disease pellagra

• Humans obtain niacin from cereals, meat, legumes

132

Oxidized, reduced forms of NAD (NADP)

133

Structure of NAD

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

134

NAD and NADP are cosubstrates for dehydrogenases

• Oxidation by pyridine nucleotides always occurs two electrons at a time

• Dehydrogenases transfer a hydride ion (H:-) from a substrate to pyridine ring C-4 of NAD+ or NADP+

• The net reaction is:

NAD(P)+ + 2e- + 2H+ NAD(P)H + H+

135

Biosynthesis of NAD(P)

136

Oxidoreductase and Dehydrogenase

137


• Oxidoreductases that transfer electron from one molecule to another

• These enzymes catalyze the oxidation reaction: A(red) + B(oxid) A(oxid) + B(red)

• In reality, free electrons do not exists as these reactions involve atoms transfer

138


• Dehydrogenases: that involve removing hydrogen from the electron donor during metabolic oxidation reactions

• Oxidases are used only for the enzymes in which the oxidation reaction with molecular oxygen (O2) participating as the electron acceptor

139

Dehydrogenase Nomenclature

• The common scheme for making names for oxidoreductases is adding donor name to the dehydrogenase, i.e. donor dehydrogenase.

• For example: alcohol dehydrogenase, lactate dehydrogenase, etc

• The proper name consists from the donor name, acceptor name together with oxidoreductase, i.e. donor: acceptor oxidoreductase

140

Dehydrogenase Nomenclature

• Sometimes the construction acceptor reductase is used: – Example: Enzyme EC 1.1.1.1

Systematic name: alcohol:NAD+

oxidoreductaseAccepted name: alcohol dehydrogenase

141

Enzymatic Classification of Dehydrogenases

• According to the Enzyme Nomenclature from NC-IUBMB the nomenclature and classification of enzymes is based on the reaction they catalyze

• Each reaction, catalyzed by enzyme is specified by the Enzyme Commission number or EC number

142

Enzymatic Classification of Dehydrogenases

• Each EC number consists of the EC and for digits separated by periods

• Each digit represents the progressively higher level of enzyme classification

• Dehydrogenases are belongs to the EC 1 Oxidoreductases group

• Oxidoreductases classification according to the substrate they utilize:

143

• EC 1.1 - Acting on the CH-OH group of donors • EC 1.2 - Acting on the aldehyde or oxo group of donors • EC 1.3 - Acting on the CH-CH group of donors • EC 1.4 - Acting on the CH-NH2 group of donors • EC 1.5 - Acting on the CH-NH group of donors • EC 1.6 - Acting on NADH or NADPH • EC 1.7 - Acting on other nitrogenous compounds as

donors • EC 1.8 - Acting on a sulfur group of donors • EC 1.9 - Acting on a heme group of donors • EC 1.10 - Acting on diphenols and related substances

as donors • EC 1.11 - Acting on a peroxide as acceptor • EC 1.12 - Acting on hydrogen as donor

144

• EC 1.13 - Acting on single donors with incorporation of molecular oxygen (oxygenases)

• EC 1.14 - Acting on paired donors, with incorporation or reduction of molecular oxygen

• EC 1.15 - Acting on superoxide as acceptor • EC 1.16 - Oxidizing metal ions • EC 1.17 - Acting on CH or CH2 groups • EC 1.18 - Acting on iron-sulfur proteins as donors• EC 1.19 - Acting on reduced flavodoxin as donor • EC 1.20 - Acting on phosphorus or arsenic in donors • EC 1.21 - Acting on X-H and Y-H to form an X-Y bond • EC 1.97 - Other oxidoreductases • EC 1.98 - Enzymes using H2 as reductant • EC 1.99 - Other enzymes using O2 as oxidant

145

Structural Classification of Dehydrogenases

• Currently, two different classifications of dehydrogenases are exists:– One is historical for polyol dehydrogenases and – Another is modern UniProt protein classification

for dehydrogenases and oxydoreductases• You still can use ancient classification, but it

is necessary to remember, that these classification are slightly different

• Please also remember, that alcohol dehydrogenase classification is slightly inconsistent

146

Dehydrogenase Catalytic Mechanism

• Dehydrogenases transfer protons to an acceptor or coenzymes such as NAD+/NADH or NADP+/NADPH, FAD/FMN

• The wide diversity of dehydrogenases does not allow to develop a uniform catalytic mechanism for all cases

• All NAD+/NADH reactions in the body involve 2 electron hydride transfers

147

• NAD+/NADH can undergo two electron redox steps, in which a hydride is transferred from a substrate to the NAD+, with the electrons flowing to the positively charged nitrogen of NAD+ which serves as an electron sink


148

149

• NADH does not react well with dioxgyen (O2)• Since single electron transfers to/from

NAD+/NADH produce free radical species which can not be stabilized effectively


150


151

Hydrogenases

• The enzymes that catalyze hydrogen production are hydrogenases (not dehydrogenses)

• Crystal structures of hydrogenases show them to be unique among metal-containing enzymes

• They contain two metals bonded to each other. The metal centers can either be both iron or one each of iron and nickel

152

Experimental Evidences for Hydride Ion Transfer

153

Alcohol Dehydrogenase

N

CONH2

R

CH3CH2OHN

CONH2

R

HH

O

C HH3C+

NAD NADH

• if run in T2O or D2O, no T or D incorporation in NADH• if run with H3CCD2OH, complete D incorporation in NADH• Results consistent with a hydride-transfer (H-) mechanism and not

a proton-transfer (H+)

N

CONH2

R

CH3CH2OHN

CONH2

R

HH

NAD NADH

C O

H

H

H3CH

Enzyme

:BC O

H3C

Enzyme

B

H

H

154

N

CONH2

R

CH3CH2OHN

CONH2

R

HH

O

C HH3C+

NAD NADH

ADH

EthanolAcetaldehyde

1. If run in T2O or D2O, no T or D incorporation in NADH

2. If run with H3CCD2OH, complete D incorporation in NADH

155

3. Results consistent with a hydride-transfer (H-) mechanism and not a proton-transfer (H+)

N

CONH2

R

CH3CH2OHN

CONH2

R

HH

NAD NADH

C O

H

H

H3CH

Enzyme

:BC O

H3C

Enzyme

B

H

H

156

Experimental Evidence for a Hydride-transfer vs an Electron-

transfer mechanism

• Cyclopropyl carbinyl radical ring opening as a probe for radical intermediates

k ~ 108 s-1

cyclopropyl carbinylradical (radical clock)

4-butenyl radical

157

Experimental Evidence for a Hydride-transfer vs an

Electron-transfer mechanism

158

CO2H

O

CO2H

O lactatedehydrogenase

lactatedehydrogenase

CO2H

OH

CO2H

OH

NADH

NADH

Product consistent with a hydride-transfer mechanism

2˚ alcohol

2˚ alcohol

159

• If an electron-transfer mechanism:

CO2H

O

CO2H

O+ e-

CO2H

O + e-

CO2H

O2 H+

CO2H

O

- keto acid

160

161

Lactate Dehydrogenase

162

Lactate Dehydrogenase

• It is a tetramer of MW 14000• It provides a good example of the occurrence

of isoenzymes• There are five forms of the enzymes can be

separated by electrophoresis• The different forms arise from five possible

way of assembling a tetramer from two types of subunits (4, 3, 22, 3 and 4)

163

Lacte Dehydrogenase Isoenzymes

LD 1 LD 2 LD 3 LD 4 LD 5

164

Lactate Dehydrogenase Isoenzymes, Cont’d

0

10

20

30

40

50

60

% Distribution

LD-1LD-2LD-3LD-4LD-5

Heart

16505

1015202530354045

% Distribution

LD-1LD-2LD-3LD-4LD-5

Skeletal Muscle

Lactate Dehydrogenase Isoenzymes, Cont’d

166

Molecular Structure of LDH

H H

H

H H

M H

H H

M MH

H M

M M

M M

M M

LD 1 LD 2 LD 3

LD4LD 5

167

LDH Isoenzymes in Liver

0

10

20

30

40

50

60

70

80

% Distribution

LD 1

LD 2

LD 3

LD 4

LD 5

168

LDH Isoenzymes in Serum

0

5

10

15

20

25

30

35

40

% Total Activity

LD-1

LD-2

LD-3

LD-4 & LD-5

169

170

LDH Assays

CH3 C

O

COOH

Pyruvate Lactate

NADH NAD

CH3 CH

OH

COOH

H+ +

171

• The NAD (colored) is bound in a bent conformation: – Only part of the LDH

enzyme is shown– The -helices are

displayed as bands, the-pleated sheets as arrows

– Amino acid side chains that are in direct contact with NAD are outlined

172

NAD Binding Domain

• (a) It consists of a 6-stranded parallel -sheet and a 4 -helix

• (b) NAD binds in an extended conformation through H bonds and salt bridges

(a)

(b)

173

The tetramer of the M4 isoenzyme

174

Active Site of LDH

• The active site of LDH showing the relative arrangement of reacting groups

• The substrate pyruvate is shown; the ‑CH3 group is replaced by ‑NH2 to form oxamate

• The hydride transfer is indicated by the bold arrow, hydrogen transfer by light arrow

175

Mechanism of Lactate Dehydrogease

176

Mechanism of Lactate Dehydrogease

Arg

N

NH

OH

C

H

CH3

B:

N

R

NH2

O

His

NAD+

+

Arg-171

-109

-195

C

O

O

Lactate

Hydride ion (H:-) is transferred from C-2 of lactate to the C-4 of

NAD+

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

177

CH3C

Lacate Dehydrogeanse

H

N

N

His

O

NADH

+N

NH2

R

HH

..

O

BH+

COO

Pyruvate

178

Ordered mechanism for lactate dehydrogenase

• Reaction of lactate dehydrogenase

• NAD+ is bound first and NADH released last

179


180

• ADH is a homodimer• Each monomer has 374 residues with molecular

weight of 74000 dalton• There are two domains:

– The NAD+-binding domain (residues 176-318) consists of a central -sheet of 6 strands flanked by helices. NAD+ binds to the C-terminus of the -sheet

– The catalytic domain (residues 1-175, 319-374) also has a / structure


181

• ADH binds two zinc ions:– One structural role– One catalytic role

• There are two Zn2+ cations per monomer, one at the catalytic site being mandatory for catalysis

• The catalytic zinc coordinates with two sulfur atoms from (3) Cys 46, Cys 174, and a nitrogen atom from His 67

• An ionizable water molecule occupies the fourth position on the zinc


182

• The fifth and final zinc coordinate is the oxygen from the alcohol

• In the active site there are three amino acids, Phe-93, Leu-57 and Leu-116, that work in concert to provide the three point binding of the alcohol substrate

• This binding accounts for the stereo-specific removal of the pro-R hydrogen


183


184


185


ADH is a homodimer

186

Reaction of ADH

187

Dehydrogenase Stereospecificity

188

STEREOCENTERSSTEREOCENTERS

One of the ways a molecule can be chiral is to have astereocenter

A stereocenter is an atom, or a group of atoms, that can potentially cause a molecule to be chiral

stereocenters cangive rise to chirality

189

BrFH

Cl

STEREOGENIC CARBONSSTEREOGENIC CARBONS

A stereogenic carbon is tetrahedral and has four different groups attached

stereocenter

(called “chiral carbons” in older literature)

HFClBr

190

Cl

ClBrBr

Cl Cl

plane ofsymmetry

side view edge view

Cl Cl

191

CONFIGURATIONCONFIGURATION

ABSOLUTE CONFIGURATION (R /S)

192

CONFIGURATIONCONFIGURATION

The three dimensional arrangement of the groups attached to an atom

Stereoisomers differ in the configuration at one ormore of their atoms

193

2

CONFIGURATIONCONFIGURATION: relates to the three dimensionalsense of attachment for groups attached to a chiralatom or group of atoms (i.e., attached to a stereocenter)

clockwise counterclockwise

(rectus) (sinister)

view with substituentof lowestpriority inback

1 2

4

3

C C

1

4

3

R S

194

DETERMINATION OF DETERMINATION OF R/S CONFIGURATIONR/S CONFIGURATIONIN FISCHER PROJECTIONSIN FISCHER PROJECTIONS

195

H

CH2OH

CHO

OH

PLACE THE PRIORITY = 4 GROUP IN ONE OF THE VERTICALPOSITIONS, THEN LOOK AT THE OTHER THREE

H

OH

OHC CH2OH1

2

3

4

1

2 3

4

R

alternatively:

H

CH2OH

CHO

OH1

2

3

4 HOCH2 CHO

OH2

1

4

3

H

R

#4 at top position

#4 at bottom position

BOTH IN BACKSAME RESULT

196

H

CH2OH

CHO

OH 1

2

3

4

FOR THE MENTALLY AGILEFOR THE MENTALLY AGILEWHY BOTHER INTERCHANGING? JUST REVERSE YOUR RESULT!

H comingtoward you

Same moleculeas on previousslide. S reverse R

Same resultas before.

197

THE SIMPLEST WAY OF ASSIGNING R/S CONFIGURATION WAS GIVEN BY EPLING (1982)

1. FIX THE PRIORITY

2. TRACE A SEMICIRCLE JOINING a b c IGNORING d

3. CLOCKWISE IS ‘R’ AND ANTICLOCKWISE ‘S’ IF ‘d’ IS VERTICAL (TOP OR BOTTOM)

4. IF ‘d’ IS ON THE HORIZONTAL LINE REVERSE THE NOTATION

198

Prochiral Center

Ethanol Acetaldehyde

199

Prochiral Center

NAD+ NADH

200

Alcohol Dehydrogenase: Pro-chirality

OH3C H

R-

R-

Pro-S face

Pro-R face

H3C

R

OHH

S 1

2

3

4

H3C

R

HOH

R 4

2

3

1

enantiomers

H3C

OH

HH

ethanol

pro-Shydrogen

pro-Rhydrogen

H3C

OH

DH

H3C

OH

HD

1

1

2

2

4

4

3

3S

R

H’s are enantiotopic,chemically equivalent

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Documents

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