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

Syllabus
• Instructor : Professor A. S. Alhomida
– Office: 2A 62; Tel: 467-5938
– E-mail: alhomida@ksu.edu.sa
– Web page: faculty.ksu.edu.sa/alhomida
• Textbook :
1. Enzyme kinetics and mechanism. Cook and Cleland, 2007
2. Enzymatic Reaction Mechanisms. Walsh, 1979
3. Introduction to Enzyme and Coenzyme Chemistry, 2 nd Edition.
Bugg, 2004
4. Contemporary Enzyme Kinetics and Mechanism. Purich, 1983
5. Structure and Mechanism in Protein Science: A guide to Enzyme
Catalysis and Protein folding . Fersht, 1999
6. Biochemistry 2 nd edition. Garrett and Grisham, Chapter, 14-16
2

Syllabus, Cont’d
7.
Lechninger's Principles of Biochemistry 4 th
Nelson and M.M. Cox., Chapter 6
8.
Biochemistry 3 rd
Chapter: 11 edition. D. L.
Edition. Mathews, Holde and Ahern.
9.
Fundamentals of Biochemistry 2 nd Edition by Voet and
Voet. Chapters: 11, 12
10. Biochemistry 3 rd Edition. Zubay. Chapters: 8-11
11. Stryer ’s Biochemistry, 5 th Edition. Berg, Tymoczko and
Stryer. Chaper 8-10.
12. Principles of Biochemistry, 4 th Edition. Horton, Scrmgeour,
Perry and Rawn. Chapter 5-7
3

Oral Presentation Project
• 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
4

Oral Presentation Project, Cont ’d
• 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 sitedirected mutagenesis or site-directed inactivation (transition state analogs)
5

Oral Presentation Project, Cont ’d
• 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 22 nd
• Submit a soft copy of your presentation saved in CD disk using PowerPoint
6

Oral Presentation Project, Cont ’d
• Completion Schedule
– On Saturday, December 22 nd , select a date for your presentation
– Before Saturday, January 5 th , 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 12 th
• The project will account for 20 out of 70 of your course grade
7

8

9

10

11

12

13

14

Glycogenolysis
Glc
Glycogen
PP a vit B
6
G1P G6P
G3P
Glycolysis
PPP
R5P
TK vit B
1
Ala
ALT vit B
6
Pyr
PDH vit B
1
,B
2
,B
3
Acetyl-CoA
Asp
AST vit B
6
OA
TCA cycle
SCoA vit B
6 a KG a KGDH vit B
1
,B
2
,B
3
Glu
15

Coenzymes and Vitamins
• Some enzymes require cofactors for activity
(1) Essential ions (mostly metal ions)
(2) Coenzymes (organic compounds)
Apoenzyme
(protein only)
(inactive)
+ Cofactor Holoenzyme
(active)
16

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
17

Types of cofactors
18

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 + , Ca 2+ , Mg 2+ )
(2) Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc, copper, cobalt )
19

20

Carbonic Anhydrase
• Carbon dioxide (CO
2
) is a major end product of aerobic metabolism
• In mammals, this CO
2 is released into the blood and transported to the lungs for exhalation
• While in the blood, CO
2 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 +
21

Carbonic Anhydrase, Cont ’d
• Almost all organisms contain enzyme, carbonic ahydrase, that catalyzes the below reaction
• Cabonic anhydrase accelerates CO
2 hydration dramatically at rate as high as k cat
=
10 6 s -1
22

Types of Carbonic Anhydrases
• a
-Carbonic anydrase:
– Found in human, animals, some bacteria and algae
– Trimer
• b
-Carbonic anydrase
– Higher plants and many bacteria and E. coli
– Has only one conserved His whereas in a has three
His
• g
-Carbonic anhydrase
– Found in bacteria Methanoscarcina thermophila
– Has three zn sites similar to acarbonic anhydrase
23

Structure of a
-Carbonic Anydrase
• Zn 2+ 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 CO
2 and bicarbonate to maintain acid-base balance in blood and other tissues and to help transport
CO
2 out of tissues
24

Structure of β-Carbonic Anhydrase
• Found in plans which is an evolutionarily distinct enzyme but participates in the same reaction and also uses a Zn 2+ in its active site
• It helps raise the concentration of CO
2 within the chloroplast to increase the carboxylation rate of the enzyme Rubisco
• It integrates CO
2 into organic carbon sugars during photosynthesis, and can only use the
CO
2 form of carbon, not carbonic acid nor bicarbonate
25

Structure of a
-Carbonic Anydrase
Three subunits
Zn bound to three His 26

Structure of g
-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
27

Human carbonic anhydrase
28

Carbonic Anhydrase, Cont ’d
• How does this Zn 2+ complex facilitates CO
2 hydration?
• A major clue comes from the pH profile of the enzymatic ally catalyzed CO2 hydration:
29

Carbonic Anhydrase, Cont ’d
• 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
30

Carbonic Anhydrase, Cont ’d
• 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 Zn 2+ -bound water molecule
• The binding of water to the positively charged
Zn 2+ center reduces the pKa of the water from
15.7 to 7
31

Carbonic Anhydrase, Cont ’d
32

Carbonic Anhydrase, Cont ’d
• The lowered pKa generates Zn 2+ -OH complex that is sufficiently nucleophilic to attack CO
2 does more readily than water
33

34

Mechanism of Carbonic
Anhydrase
His
His
His
Zn
2+
B:
H
O
CO
2
His
His
His
Zn
2+
B:
H
O
O C O
35

Mechanism of Carbonic
Anhydrase, Cont ’d
• Zn 2+ ion promotes the ionization of bound
H
2
O. Resulting nucleophilic OH attacks carbon of CO
2
• The pKa of water drops from 15.7-7 when it is coordinate to Zn 2+
• HO is 4 orders of magnitude more nucleophlic than is water
36

His
His
His
Zn
2+
B:
H
O
C
O O
H
2
O
His
His
His
Zn
2+
B :
O
H
O
C
O
H
O
H
37

His
His
His
Zn
2+
H
O
O
C
O
B H
H
O
Tetrahedral intermediate
His
His
His
Zn
2+
H
O
B H
O
HO C O
Bicarbonate
38

Mechanism of Carbonic
Anhydrase, Cont ’d
1. The important of Zn 2+ -OH comlpex suggests a simple mechanism of CO2 hydration:
2. Zn 2+ facilitates the release of a H + from water, which generates a OH -
3. The CO
2 binds to the enzyme ’s active site and is positioned to react with the OH -
39

Mechanism of Carbonic
Anhydrase, Cont ’d
3. The OH attacks nucleophilically CO
2 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
40

Iron in Metalloenzymes
• Iron undergoes reversible oxidation and reduction:
Fe 3+ + e (reduced substrate) Fe 2+ + (oxidized substrate)
• Enzyme heme groups and cytochromes contain iron
41

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
42

Iron-sulfur clusters
• Iron atoms are complexed with an equal number of sulfide ions (S 2) and with thiolate groups of Cys side chains
43

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
44

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)
45

46

Metabolite Coenzymes
• Nucleoside triphosphates are examples
5`-C
ATP g b a
47

Reactions of ATP
• ATP is a versatile reactant that can donate its:
(1) Phosphoryl group ( g
-phosphate)
(2) Pyrophosphoryl group ( g , b phosphates)
(3) Adenylyl group (AMP)
(4) Adenosyl group
48

• 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)
49

50

51

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
52

Alkylation Reactions, Cont ’d
53

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
54

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 b
-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
55

Enamines, Cont ’d
56

Aldol Reactions
• Aldol Reactions Require Several Levels of
Control:
• Enol versus carbonyl component:
• carbonyl compounds with acidic a
-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
57

Aldol Reactions, Cont ’d
• 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
58

Aldol Reactions, Cont ’d
• In biological systems the regioselectivity is controled by the steric environment of the enzyme active site
59

Aldol Reactions, Cont ’d
• 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
60

61

SAM Biosynthesis
• ATP is a source of other metabolite coenzymes such as S-adenosylmethionine
(SAM)
• SAM donates methyl groups in many biosynthesis reactions
Methionine + ATP SAM + P i
+ PP i
62

Structure of SAM
• Activated methyl group in red
63

Functions of SAM
1. SAM donates the methyl group for many methylation reactions: Methylation of norepinephrins
64

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
65

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 b
-Lys
5. Lys 2,3-aminomustase catalyzes the reaction by 1,2 rearrangement mechanism similar to Vit
B
12
-dependent mutase, but didn ’t use Vit B
12 instead required PLP and SAM for activity and a reduced [4Fe4S] cluster
66

SAM as Methyl Group Donor
– Methylation of bases in tRNA
– Methylation of cytosine residues in DNA
– Methylation of norepinephrine
67

SAM Cycle
1. SAM synthase (Met adenosyl transferase)
2. Methyltransferase
3. S-adenosyl homocysteinase
4. Homocysteine methyltransferase
68

69

Mechanism of SAM Synthase
H
3
N
H
C COO
CH
2 2
S
:
CH
3
Methionine
Unusual displacement of triphosphate reaction
O
O
P
O
O P
O
O P
O
Nucleophilic attack
(S
N
2 mechanism)
O O
O
NH
2
N
N
N
N
CH
2
O
H
H
H
H
OH OH
ATP
70

Mechanism of SAM Synthase,
Cont ’d
• 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 (PPP formed i
) is
71

Mechanism of SAM Synthase
Supernucleophile
SAM synthase
H
3
N
O O O
O P
O
O P
O
O P
O
O
PPP i
H
2
O
H
C COO
CH
2 2
S
CH
3
NH
2
N
N
N
N
CH
2
O
H
H
H
H
OH OH
SAM
O O
O
O P
O
O P
O
PP i
O
O P
O
O
O
2 O P
O
O
P i
Very good leaving group because of positively charged of
S atom
72

Mechanism of SAM Synthase,
Cont ’d
• PPP i is then hydrolyzed by the same enzyme into PP i and P i 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
73

Mechanism of SAM Synthase,
Cont ’d
• The other being the formation of adenosylcobalamin
• The hydrolysis of PPP i drives the reaction to right highly exergoic in the synthetic direction
74

75

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
76

SAM-Dependent Methyltransferase,
Cont ’d
• Human thiopurine Smethyltransferase
(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, 6thioguanine
77

SAM-Dependent Methyltransferase,
Cont ’d
• All methylation reactions requiring SAM are simple S
N
2 (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
78

SAM-Dependent Methyltransferase,
Cont ’d
• This is accomplished through the action of Sadenosylhomocysteinase that converts SAH into adenosine and homocysteine
• Homocysteine is converted into Met and adenosine (Ado) into inosine (via SAM cycle)
79

Mechanism of SAM-Dependent
Methyltransferase
80

Mechanism of SAM-Dependent
Methyltransferase
HO CH
2
CH
2
..
2
OH
HO
Norepinephrine
Nucleophilic attack (S
N
2
Mechanism)
H
3
N
H
C COO
N
CH
2 2
NH
2
N
S
CH
3
SAM
N
N
CH
2
H
O
H H
OH OH
H
81

Mechanism of SAM-Dependent
Methyltransferase, Cont ’d
HO
HO
CH
3
CH
2
CH
2
NH
2
OH
Epinephrine
+
H
3
N
H
C COO
N
CH
2 2
NH
2
N
S
N
N
CH
2
O
H
H
H
H
OH OH
S-adenosylhomocysteine (SAH)
82

83

SAM-Dependent Radical Enzymes
• Organic radicals are used by a number of enzymes to catalyze biochemical transformations with high-energy barriers that would be difficult to accomplish through nonradical 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
84

SAM-Dependent Radical Enzymes
• Organic radicals can be generated in enzymes through only three general mechanisms:
– Metal-activated oxygen biochemistry
– Adenosylcobalamin (Vit B
12 or
) biochemistry,
– Reduction of the sulfonium of SAM
85

(Formate C-Acetyltransferase)
86

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
87

Structure of Pyruvate Formate Lyase
• It is a homodimer made of 85 kD, 759-residue subunits
• It has a 10-stranded b
/ a barrel motif into which is inserted a b 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
88

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
89

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
90

SAM-[4Fe4S] Cluster
SAM
• The interaction of SAM with the [4Fe –4S] 1+ of activated en\yme
• a
-N and a
-carboxyl O of Met anchors the SAM to the cluster with the sulfonium interacting with a sulfide from the cluster a
[4Fe4S] cluster
91

Regulationn of Pyruvate Formate
Lyase
Radical
(AE) Activase
Radical Gly-734
(DE) Deactivase
92

Reaction of Pyruvate Formate Lyase
H
N
734
O
N
H
H H
Gly-734
Pyruvate formate lyase
[4Fe4S] red
+
SAM
H
N
734
O
N
H
Gly-734 radical
93

Reaction of Pyruvate Formate Lyase,
Cont ’d
Pyruvate formate lyase
O
H
3
C
O
Pyruvate
O
H
N
734
O
N
H
Gly-734 radical
O
O
H
Formate
O
H
3
C
Acetyl-CoA
94

95

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
96

Generation of 5`-deoxyadenosyl
Radical from SAM by [4Fe4S] Cluster
Ad
O
Enz
Fe
S
S
H
3
C
S
Fe
S
Fe
Fe S
H
N
2
O
H
OH
O
SAM
OH
2
5`-deoxyadenosyl radical
Ad
O
Enz
Fe
S
S
H
3
C
S
Fe
H
2
C
S
H
N
2
Fe
O
Fe S
O
H
OH
OH
97

98

Mechanism of Pyruvate Formate
Lyase
Gly-734
Cys-419
Cys-418
Radical transfer from Gly-734 to
Cys-419
H
99

Mechanism of Pyruvate Formate
Lyase, Cont ’d
Gly-734
H H
S
Cys-419
Cys-418
Radical transfer from Cys-419 to
Cys-418
Pyruvate
Gly-734
H H
O
Cys-419
S
O
Cys-418
H
3
C
O
100

Mechanism of Pyruvate Formate
Lyase, Cont ’d
Gly-734
H
Cys-419
H
H
3
C
O
S
O
O
Cys-418
Tetrahedral radical intermediate
Gly-734
Cys-419
H H
O S
Cys-418
O formate radical intermediate
H
3
C
O
Thioester
(acyl-enzyme)
101

Mechanism of Pyruvate Formate
Lyase, Cont ’d
Gly-734
Radical transfer from Cys-419 to
CoA
CoA-S H
H
Cys-419
H H
S
O
CoA-S H
H
3
C
S
O
Cys-418
O
Formate
102

Mechanism of Pyruvate Formate
Lyase, Cont ’d
Gly-734
Cys-419
H H
CoA-S
Radical transfer from CoA to acetate
H
3
C
S
O
Cys-418
Gly-734
H
Cys-419
H
CoA-S
S
H
3
C
O
Cys-418
Tetrahedral radical intermediate
103

Mechanism of Pyruvate Formate
Lyase, Cont ’d
Gly-734
O
CoA-S
C H
3
Acetyl-CoA
Cys-419
H H
S
Radical Cys-418
Cys-418
104

Mechanism of Pyruvate Formate
Lyase, Cont ’d
Gly-734
H H
Cys-419
S
Cys-418 e
Cys-418 radical enzyme
Gly-734
H H
Cys-419
Cys-418
Cys-418 radical inactivated enzyme
105

Mechanism of Pyruvate Formate
Lyase, Cont ’d
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 acetylenzyme intermediate (which now contains the radical)
3. The acetyl-enzyme intermediate releases a formyl radical that undergoes hydrogenatom transfer with Cys-419
106

Mechanism of Pyruvate Formate
Lyase, Cont ’d
4. CoA comes in and undergoes hydrogenatom 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
107

Mechanism for Generating Radical
Gly-734
From favorodoxin
Trasfer radical to inactivated
Gly-724 enzyme
108

Mechanism for Generating Radical Gly-
734
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 a
-nitrogen and a
carboxyl oxygen to anchor SAM in the active site
109

Mechanism for Generating Radical Gly-
734, Cont ’d
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 innersphere electron transfer from the bridging sulfide to the sulfonium of SAM, and facilitates homolytic bond cleavage and creation of the adenosyl radical
110

Mechanism for Generating Radical Gly-
734, Cont ’d
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
111

112

• 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
113

114

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
115

Vitamin C (ascorbic acid) in Foods
116

117

Niacin in Foods
118

Niacin in Foods
119

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
120

Reduction Reactions, Cont ’d
121

Reduction Reactions, Cont ’d
• The pyridinium ring acts as hydride acceptor in the oxidation step, whilst 1,4dihydropyridine system acts as hydride donor in the reduction step:
122

Reduction Reactions, Cont ’d
• 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
123

Reduction Reactions, Cont ’d
124

Reduction Reactions, Cont ’d
• 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)
125

Oxidation Reactions
• 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
126

Oxidation Reactions, Cont ’d
• In fact, NAD and NADH have to be reversible redox pairs to allow the coenzyme and the enzyme to act as true catalysts
127

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
128

Cytochrome-P450-dependant
Enzymes, Cont ’d
129

Non-Hem a
-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
130

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
131

132

133

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 +
134

Biosynthesis of NAD(P)
135

136

Oxidoreductase and Dehydrogenase
• 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
137

Oxidoreductase and Dehydrogenase
• 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 (O
2
) participating as the electron acceptor
138

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
139

Dehydrogenase Nomenclature
• Sometimes the construction acceptor reductase is used:
– Example: Enzyme EC 1.1.1.1
Systematic name: alcohol:NAD + oxidoreductase
Accepted name: alcohol dehydrogenase
140

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
141

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:
142

• 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
143

• 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
144

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
145

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
146

Dehydrogenase Catalytic
Mechanism
• 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
147

148

Dehydrogenase Catalytic
Mechanism
• NADH does not react well with dioxgyen (O
2
)
• Since single electron transfers to/from
NAD + /NADH produce free radical species which can not be stabilized effectively
149

Dehydrogenase Catalytic
Mechanism
150

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
151

152

CONH
2
Alcohol Dehydrogenase
H H
CONH
2
CH
3
CH
2
OH
N
R
NADH
+
H
3
C
O
C H
N
R
NAD
• if run in T
2
O or D
2
O, no T or D incorporation in NADH
• if run with H
3
CCD
2
OH, complete D incorporation in NADH
• Results consistent with a hydride-transfer (H ) mechanism and not a proton-transfer (H + )
Enzyme
Enzyme
H
3
C
H
C O
H
:B
H
3
C
H
C O
H B
H
H H
CONH
2 CONH
2
CH
3
CH
2
OH
N
R
NAD
N
R
NADH
153

H H
CONH
2 CONH
2 ADH
H
3
C
O
C H
CH
3
CH
2
OH +
N
R
N
R
Acetaldehyde
Ethanol
NADH
NAD
1. If run in T
2
O or D
2
O, no T or D incorporation in
NADH
2. If run with H
3
CCD
2
OH, complete D incorporation in NADH
154

3. Results consistent with a hydride-transfer (H ) mechanism and not a proton-transfer (H + )
H
3
C
H
C O
H
Enzyme
:B
H
CONH
2
N
R
NAD
CH
3
CH
2
OH
H
3
C
H
C O
H
Enzyme
B
H H
CONH
2
N
R
NADH
155

Experimental Evidence for a Hydride-transfer vs an Electrontransfer mechanism
• Cyclopropyl carbinyl radical ring opening as a probe for radical intermediates k ~ 10
8
s
-1 cyclopropyl carbinyl radical (radical clock)
4-butenyl radical
156

157

O lactate dehydrogenase
OH
CO
2
H
NADH
CO
2
H
2 ˚ alcohol
O lactate dehydrogenase
OH
CO
2
H
NADH
CO
2
H
2 ˚ alcohol
Product consistent with a hydride-transfer mechanism
158

• If an electron-transfer mechanism:
O
CO
2
H
+ e
-
O
CO
2
H
O
CO
2
H
2 H
+
O
CO
2
H a
- keto acid
O
CO
2
H
+ e
-
159

160

161

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 ( a
4
, a
3 b
, a
2 b
2
, ab
3 and b
4
)
162

LD 1 LD 2 LD 3 LD 4 LD 5
163

Heart
60
50
40
%
Distribution
30
20
10
0
LD-1
LD-2
LD-3
LD-4
LD-5
164

Skeletal Muscle
% 25
Distribution 20
15
10
5
0
45
40
35
30
LD-1
LD-2
LD-3
LD-4
LD-5
165

LD 1
LD 2 LD 3
H H
H H H H
H H
M H M M
M M
M M
LD 5
H M
M M
LD4
166

80
70
60
%
Distribution
50
40
30
20
10
0
LD 1
LD 2
LD 3
LD 4
LD 5
167

% Total
25
20
Activity
15
10
5
0
40
35
30
LD-1
LD-2
LD-3
LD-4 & LD-5
168

169

Pyruvate
O
CH
3
C COOH
Lactate
OH
CH
3
CH COOH
H + + NADH
NAD
170

• The NAD (colored) is bound in a bent conformation:
– Only part of the LDH enzyme is shown
– The a
-helices are displayed as bands, the b
pleated sheets as arrows
– Amino acid side chains that are in direct contact with NAD are outlined
171

NAD Binding Domain
• (a) It consists of a 6stranded parallel b
sheet and a 4 a
helix
• (b) NAD binds in an extended conformation through H bonds and salt bridges
(a)
(b)
172

The tetramer of the M4 isoenzyme
173

Active Site of LDH
• The active site of LDH showing the relative arrangement of reacting groups
• The substrate pyruvate is shown; the -CH
3 group is replaced by -NH
2 oxamate to form
• The hydride transfer is indicated by the bold arrow, hydrogen transfer by light arrow
174

175

Mechanism of Lactate Dehydrogease
Arg-171
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?
O
O
CH
3
C
C
H
H
O
Lactate
O
Arg -109
His -195
N
N
H
B:
+
N
R
NAD
+
NH
2
176

Lacate Dehydrogeanse
O
CH
3
C COO
Pyruvate
+
H H
O
..
N
R
NADH
NH
2
H
N
N
His
BH
+
177

Ordered mechanism for lactate dehydrogenase
• Reaction of lactate dehydrogenase
• NAD + is bound first and NADH released last
178

179

Alcohol Dehydrogenase
• 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 b
-sheet of 6 strands flanked by a helices. NAD + binds to the C-terminus of the b
-sheet
– The catalytic domain (residues 1-175, 319-374) also has a a
/ b structure
180

Alcohol Dehydrogenase
• ADH binds two zinc ions:
– One structural role
– One catalytic role
• There are two Zn 2+ 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
181

Alcohol Dehydrogenase
• 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 stereospecific removal of the proR hydrogen
182

Alcohol Dehydrogenase
183

Alcohol Dehydrogenase
184

Alcohol Dehydrogenase
ADH is a homodimer
185

Reaction of ADH
186

187

One of the ways a molecule can be chiral is to have a stereocenter
A stereocenter is an atom, or a group of atoms, that can potentially cause a molecule to be chiral stereocenters can give rise to chirality
188

(called “chiral carbons” in older literature)
Cl
H
Br
F
A stereogenic carbon is tetrahedral and has four different groups attached
189
H
F
Cl
Br

plane of symmetry
Cl
Cl
Br side view
Cl
Cl
Cl
Br
Cl

ABSOLUTE CONFIGURATION (R /S)
191

The three dimensional arrangement of the groups attached to an atom
Stereoisomers differ in the configuration at one or more of their atoms
192

CONFIGURATION : relates to the three dimensional sense of attachment for groups attached to a chiral atom or group of atoms (i.e., attached to a stereocenter) clockwise
1
4
C view with substituent of lowest priority in back
2
3
(rectus)
2
1 counter clockwise
C
4
3
(sinister)
193

194

PLACE THE PRIORITY = 4 GROUP IN ONE OF THE VERTICAL
POSITIONS, THEN LOOK AT THE OTHER THREE
2
CHO
OH
1
4
H
CH
2
OH
3 alternatively:
4
H
2
CHO
OH
1
CH
2
OH
3
H
4
#4 at top position
OHC
2
3
R
HOCH
2
CH
2
OH
OH
1
R
3
BOTH IN BACK
SAME RESULT
1
OH
2
CHO
4
H

WHY BOTHER INTERCHANGING?
JUST REVERSE YOUR RESULT!
Same molecule as on previous slide.
4
H
2
CHO
S
OH
1 reverse R
Same result as before.
CH
2
OH
3
H coming toward you
196

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
197

Ethanol
Prochiral Center
Acetaldehyde
198

NAD +
Prochiral Center
NADH
199

Alcohol Dehydrogenase: Pro-chirality
R
-
ProS face
H
3
C
O
H
R
-
ProR face ethanol
OH
H
3
C
H
H proS hydrogen proR hydrogen
H
3
C
2
S
3
R
1
OH
H
4 enantiomers
2
H
3
C
R
3
R
1
OH
H 4
2
H
3
C
1
OH
R
H
4
D
3
2
H
3
C
1
OH
4
S
3
D
H
H’s are enantiotopic, chemically equivalent
200

201