Transcript Vitamins and Coenzymes - KSU - Home

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: [email protected]
– 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, 2nd 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 2nd edition. Garrett and Grisham, Chapter, 14-16
2
Syllabus, Cont’d
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: 8-11
11. 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
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 22nd
• Submit a soft copy of your presentation saved
in CD disk using PowerPoint
6
Oral Presentation Project, Cont’d
• 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
7
8
Vitamins and Coenzymes
9
10
11
12
13
14
Vitamins in Metabolic
Pathways
Glycogenolysis
Glc
PP a
vit B6
G1P
Glycogen
Ala
Asp
Glycolysis
PPP
G6P
ALT
vit B6
R5P
TK
vit B1
G3P
Pyr
PDH
vit B1,B2,B3
Acetyl-CoA
AST
vit B6
OA
TCA
cycle
SCoA
aKG
vit B6
aKGDH
vit B1,B2,B3
Glu
15
Coenzymes and Vitamins
• Some enzymes require cofactors for activity
(1) Essential ions (mostly metal ions)
(2) Coenzymes (organic compounds)
Apoenzyme + Cofactor
(protein only)
Holoenzyme
(active)
(inactive)
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+, Ca2+, Mg2+)
(2) Metalloenzymes contain firmly bound metal
ions at the enzyme active sites (examples:
iron, zinc, copper, cobalt )
19
Carbonic Anhydrase
20
Carbonic Anhydrase
• 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+
21
Carbonic Anhydrase, Cont’d
• 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
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 a-carbonic anhydrase
23
Structure of a-Carbonic Anydrase
• Zn2+ is coordinated by the imidazole rings of
three His residues, His-94, His-96 and His119
• 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
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 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
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 Zn2+ complex facilitates CO2
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 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
31
Carbonic Anhydrase, Cont’d
32
Carbonic Anhydrase, Cont’d
• The lowered pKa generates Zn2+-OHcomplex that is sufficiently nucleophilic
to attack CO2 more readily than water
does
33
Mechanism of Carbonic
Anhydrase
34
Mechanism of Carbonic
Anhydrase
His
His
His
His
His
Zn2+
His
Zn2+
B:
O
B:
O
H
H
O
C
O
CO2
35
Mechanism of Carbonic
Anhydrase, Cont’d
• 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
36
His
His
His
His
His
His
Zn2+
Zn2+
B:
O
O
H
H
O
H
C
H2O
C
O
B:
O
O
H
O
37
His
His
His
His
His
Zn2+
Zn2+
BH
O
H
O
O
O
C
BH
H
H
O
Tetrahedral
intermediate
His
O
HO C O
Bicarbonate
38
Mechanism of Carbonic
Anhydrase, Cont’d
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 OH3. The CO2 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 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
40
Iron in Metalloenzymes
• Iron undergoes reversible oxidation and
reduction:
Fe3+ + e- (reduced substrate)
Fe2+ + (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 (S2-) 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
Metabolite Coenzymes
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
Carbon-Carbon Bond
Formation
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
S-Adenosylmethionine
(SAM)
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 + Pi + PPi
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
B12-dependent mutase, but didn’t use Vit B12
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
Mechanism of SAM Synthase
(Met Adenosyl Transferase)
69
Mechanism of SAM Synthase
Unusual displacement of
triphosphate reaction
NH2
H
N
N
H 3N
C
CH2
COO
O
O
2
O
P O P O P
O
O
N
N
O
CH2
O
H
S:
CH3
O
O
H
H
Nucleophilic attack
(SN2 mechanism)
H
OH OH
ATP
Methionine
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 (PPPi ) is
formed
71
Mechanism of SAM Synthase
Supernucleophile
Very good
leaving
group
because of
positively
charged of
S atom
H
H3N
NH2
COO
C
N
N
SAM synthase
CH2
2
N
N
S
CH2
H
CH3
O
H
H
H
OH OH
SAM
O
O
O
O
P O P O P
O
O
PPPi
O
O
O
O
P O P
O
H 2O
O
O
PPi
O
O
O
O
2
O
P O
O
Pi
P O
O
72
Mechanism of SAM Synthase,
Cont’d
• 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
73
Mechanism of SAM Synthase,
Cont’d
• The other being the formation of
adenosylcobalamin
• The hydrolysis of PPPi drives the reaction to
right highly exergoic in the synthetic direction
74
SAM-Dependent
Methyltransferase
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 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
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
H
H3N
HO
CH2CH2N
..H2
HO
OH
NH2
COO
C
N
CH2
2
S
N
N
CH2
Norepinephrine
Nucleophilic
attack (SN2
Mechanism)
N
CH3
SAM
O
H
H
H
H
OH OH
81
Mechanism of SAM-Dependent
Methyltransferase, Cont’d
H
H3N
CH3
HO
CH2CH2NH2
HO
OH
Epinephrine
C
NH2
COO
N
N
+
CH2
S
2
N
N
CH2
H
O
H
H
H
OH OH
S-adenosylhomocysteine (SAH)
82
SAM-Dependent Radical
Enzymes
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 B12) biochemistry,
or
– Reduction of the sulfonium of SAM
85
Pyruvate Formate Lyase
(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 Gly734 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 Ala272), 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
Radical Gly-734
(AE) Activase
(DE) Deactivase
92
Reaction of Pyruvate Formate Lyase
H
O
N 734
H
H
Gly-734
N
Pyruvate
formate lyase
H
H
N 734
O
N
H
[4Fe4S]
red
+
SAM
Gly-734 radical
93
Reaction of Pyruvate Formate Lyase,
Cont’d
Pyruvate
formate lyase
H
O
N 734
O
Gly-734 radical
N
H
O
O
H
H3C
O
CoA
O
Formate
O
Pyruvate
H3C
S
Acetyl-CoA
CoA
94
Mechanism of Pyruvate
Formate Lyase
95
Role of Catalytic Residues
Gly-734 (glycyl radical)
– Transfers the radical on and off Cys-418, via Cys419
• 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
5`-deoxyadenosyl radical
2
Ad
Ad
O
O
Enz
OH
H3C
S
Fe
S
S
S
H
S
H3C
S
OH
Fe
S
O
O
SAM
OH
H2C
Fe
Fe
Fe
H2
N
Enz
S
S
H
OH
Fe
O
Fe
Fe
H2
N
O
S
97
Mechanism of Pyruvate
Formate Lyase
98
Mechanism of Pyruvate Formate
Lyase
Gly-734
Cys-419
H
Radical transfer
from Gly-734 to
Cys-419
Cys-418
S
H
S
99
Mechanism of Pyruvate Formate
Lyase, Cont’d
Gly-734
Gly-734
Cys-419
H
H
S
H
Cys-419
Cys-418
H
H
H
S
Cys-418
S
O
O
Pyruvate
Radical transfer
from Cys-419 to
Cys-418
S
H3C
O
100
Mechanism of Pyruvate Formate
Lyase, Cont’d
Gly-734
Gly-734
Cys-419
Cys-419
H
H
H
S
H
H
H
Cys-418
Cys-418
O
S
O
S
S
H3C
O
O
Tetrahedral radical
intermediate
O
H3C
formate radical
intermediate
O
Thioester
(acyl-enzyme)
101
Mechanism of Pyruvate Formate
Lyase, Cont’d
Gly-734
Cys-419
Radical transfer
from Cys-419 to
CoA
S
H
H
Cys-418
S
CoA-S
H
H3C
CoA-S H
O
O
H
O
Formate
102
Mechanism of Pyruvate Formate
Lyase, Cont’d
Gly-734
Gly-734
Cys-419
H
H
H
Cys-419
S
Cys-418
H
H
H
S
Cys-418
S
S
CoA-S
CoA-S
Radical transfer
from CoA to
acetate
H3C
O
H3C
O
Tetrahedral radical
intermediate
103
Mechanism of Pyruvate Formate
Lyase, Cont’d
Gly-734
Cys-419
H
H
H
S
Cys-418
S
O
CoA-S
Radical Cys-418
CH3
Acetyl-CoA
104
Mechanism of Pyruvate Formate
Lyase, Cont’d
Gly-734
Gly-734
Cys-419
H
H
H
Cys-419
S
H
Cys-418
S
Cys-418 radical enzyme
e
H
H
S
Cys-418
H
S
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 Gly734
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 acarboxyl oxygen to anchor SAM in the active
site
109
Mechanism for Generating Radical Gly734, 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 Gly734, 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
Vitamin-Derived Coenzymes
112
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
113
Vitamin C
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
Nicotinamide Adenine
Dinucleotide
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
Oxidized, reduced forms of NAD (NADP)
132
Structure of NAD
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
Oxidoreductase and
Dehydrogenase
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 (O2) 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 (O2)
• 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
Experimental Evidences for
Hydride Ion Transfer
152
Alcohol Dehydrogenase
H H
CONH2
CONH2
CH3CH2OH
N
N
R
R
O
+
H3C C H
NADH
NAD
• 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+)
Enzyme
Enzyme
H B
H3C
:B
H
C O
H
H
H3C C O
H
H H
CONH2
CONH2
CH3CH2OH
N
N
R
R
NAD
NADH
153
H H
CONH2
ADH
CH3CH2OH
N
R
NAD
CONH2
Ethanol
N
R
O
+
H3C C H
Acetaldehyde
NADH
1. If run in T2O or D2O, no T or D incorporation in
NADH
2. If run with H3CCD2OH, complete D incorporation
in NADH
154
3. Results consistent with a hydride-transfer (H-)
mechanism and not a proton-transfer (H+)
Enzyme
Enzyme
H B
H3C
:B
H
C O
H
H
H3C C O
H
H H
CONH2
CONH2
CH3CH2OH
N
N
R
R
NAD
NADH
155
Experimental Evidence for
a Hydride-transfer vs an Electrontransfer mechanism
• Cyclopropyl carbinyl radical ring
opening as a probe for radical
intermediates
k ~ 108 s-1
cyclopropyl carbinyl
radical (radical clock)
4-butenyl radical
156
Experimental Evidence for
a Hydride-transfer vs an
Electron-transfer mechanism
157
lactate
dehydrogenase
O
CO2H
NADH
OH
CO2H
2˚ alcohol
lactate
dehydrogenase
O
OH
CO2H
CO2H
NADH
2˚ alcohol
Product consistent with a hydride-transfer
mechanism
158
• If an electron-transfer mechanism:
+ e-
O
O
CO2H
CO2H
O
2 H+
CO2H
+ e-
O
CO2H
O
CO2H
a- keto acid
159
160
Lactate Dehydrogenase
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 (a4, a3b, a2b2, ab3 and b4)
162
Lacte Dehydrogenase
Isoenzymes
LD 1
LD 2
LD 3
LD 4
LD 5
163
Lactate Dehydrogenase
Isoenzymes, Cont’d
Heart
60
50
40
%
30
Distribution
20
LD-1
LD-2
LD-3
LD-4
LD-5
10
0
164
Lactate Dehydrogenase
Isoenzymes, Cont’d
Skeletal Muscle
45
40
35
30
25
%
Distribution 20
15
10
5
0
LD-1
LD-2
LD-3
LD-4
LD-5
165
Molecular Structure of LDH
LD 1
LD 2
LD 3
H
H
H
H
H
H
H
H
M
H
M
M
M
M
H
M
M
M
M
M
LD 5
LD4
166
LDH Isoenzymes in Liver
80
70
60
50
%
40
Distribution
30
20
10
0
LD 1
LD 2
LD 3
LD 4
LD 5
167
LDH Isoenzymes in Serum
40
35
30
25
% Total
20
Activity
15
10
LD-1
LD-2
LD-3
LD-4 & LD-5
5
0
168
169
LDH Assays
Pyruvate
O
CH3
C
COOH
H+ + NADH
Lactate
OH
CH3 CH
COOH
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 bpleated 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 bsheet and a 4 ahelix
• (b) NAD binds in an
extended
conformation
through H bonds
and salt bridges
(b)
(a)
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 -CH3
group is replaced
by -NH2 to form
oxamate
• The hydride transfer is
indicated by the bold
arrow, hydrogen
transfer by light arrow
174
Mechanism of Lactate
Dehydrogease
175
Mechanism of Lactate Dehydrogease
Arg-171
Arg -109
Hydride ion (H:-) is
transferred from
C-2 of lactate to the C-4 of
NAD+
O
His-195
H
CH3 C
O C
N
H
O
Lactate
N
H
O
B:
NH2
Electron sink (Stored 2 electrons
and one H+). Source & Where?
+
N
R
NAD+
176
Lacate Dehydrogeanse
O
CH3C COO
Pyruvate
H H
+
..
O
NH2
N
R
NADH
His
H
N
N
BH+
177
Ordered mechanism for
lactate dehydrogenase
• Reaction of lactate dehydrogenase
• NAD+ is bound first and NADH released last
178
Alcohol Dehydrogenase
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 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
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 pro-R hydrogen
182
Alcohol Dehydrogenase
183
Alcohol Dehydrogenase
184
Alcohol Dehydrogenase
ADH is a homodimer
185
Reaction of ADH
186
Dehydrogenase
Stereospecificity
187
STEREOCENTERS
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
STEREOGENIC CARBONS
(called “chiral carbons” in older literature)
Cl
H
stereocenter
F
Br
A stereogenic carbon is tetrahedral
and has four different groups attached
189
H
F
Cl
Br
plane of
symmetry
Cl
Cl
Br
Cl
Cl
Cl
Br
Cl
side view
edge view
190
CONFIGURATION
ABSOLUTE CONFIGURATION (R /S)
191
CONFIGURATION
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
2
2
C
counter
clockwise
C
4
4
3
view with
substituent
of lowest
priority in
back
1
R
3
(rectus)
S
(sinister)
193
DETERMINATION OF
R/S CONFIGURATION
IN FISCHER PROJECTIONS
194
PLACE THE PRIORITY = 4 GROUP IN ONE OF THE VERTICAL
POSITIONS, THEN LOOK AT THE OTHER THREE
2
4
CHO
4
H
OH
1
CH2OH
OHC
2
CH2OH
OH R 3
3
alternatively:
1
CHO
4
OH 1
CH2OH
3
BOTH IN BACK
SAME RESULT
1
2
H
#4 at top position
H
OH
R
3
2
CHO
HOCH2
4
H
195
#4 at bottom position
FOR THE MENTALLY AGILE
WHY BOTHER INTERCHANGING?
JUST REVERSE YOUR RESULT!
Same molecule
as on previous
slide.
2
CHO
4
H
S
OH
1
reverse
R
Same result
as before.
CH2OH
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
IGNORING d
b
c
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
Prochiral Center
Ethanol
Acetaldehyde
198
Prochiral Center
NAD+
NADH
199
Alcohol Dehydrogenase: Pro-chirality
R-
3
R
Pro-S face
O
H3C
1
OH
H
4
S
H3C
2
H
enantiomers
Pro-R face
R-
1
OH
H 4
2
H3C
R
R
3
1
OH
OH
ethanol
pro-R
H hydrogen
H3C
H
pro-S
hydrogen
2
H3C R
3
D
H
4
1
OH
2
H3C S
D
3
H’s are enantiotopic,
chemically equivalent
4
H
200
201