Ch. 3: Vitamins, Cofactors, Coenzymes, Enzymes
∗ Readings
Ø P. J. Bruice, Organic Chemistry, 2nd ed., Prentice-Hall, Upper Saddle
River, New Jersey, 1998, Chapter 22.
Ø H. Dugas, Bioorganic Chemistry - A Chemical Approach to Enzyme
Action, 3rd ed., Springer Verlag, New York, 1996, chapter 7.
Ø O. Isler, G. Brubacher, Vitamins I: Fat-Soluble Vitamins, Thieme
Verlag, Stuttgart, 1982.
Ø O. Isler, G. Brubacher, S. Ghisla, B. Kraeutler, Vitamins II: WaterSoluble Vitamins, Georg Thieme Verlag, Stuttgart, 1988.
Ø http://www.indstate.edu/thcme/mwking/vitamins.html
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Glossary
• vitamin (water soluble vitamin, fat soluble vitamin): a substance
•
•
•
•
•
•
needed in small amounts for normal body functions that the body
cannot synthesize in adequat amounts
enzyme: a protein that is a catalyst
metalloenzyme: an enzyme that has a tightly bound metal ion
cofactor: an organic molecule or a metal ion that certain enzymes
need in order to catalyze a reaction or a process
coenzyme: an organic molecule (vitamin) as cofactor
prosthetic group: a cofoctor permanently associated with the
protein, often covalently bound
holoenzyme: catalytically active enzyme-cofactor complex.
apoenzyme: an enzyme without its cofactor (enzymatically inactive
protein)
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Glossary
• metabolism: reactions that living organism carry out
• catabolism: energy and simple molecules are formed from
complex molecules
• anabolism: synthesis of complex biomolecules using energy
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Problems - 1
@ Vitamin C and Vitamin E are both natural radical inhibitors. Explain why
this is so.
@ Explain why vitamin C is a „strong“ acid
@ Ascorbinsäure can be methylated by diazomethane. Which functional
group is most sensitive towards methylation?
@ (L)-Sorbose which is an intermediate in the technical synthesis of vitamin
C, forms a furanosid by reaction with aceton/H+.
Discuss the mechanism of formation. Are there diastereotopic methyl
groups?
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Problems - 2
@ Apply Woodward-Hoffmann-Rules for the synthesis of Vitamin D3.
Sum up the WH-rules for the electrocyclic and sigmatropic reactions
under thermal respectively photochemical conditions.
a) Which stereoisomer is formed by irradiating (2E,4Z,6Z)octatriene?
@ Stereoselective oxidoreduction of ketones and alcohols using horse
liver alcohol dehydrogenase (HLADH) needs NADH as cofactor.
NADH is rather expensive and has to be recycled. Recyclization can
be achieved by natrium dithionite. Formulate the reaction sequence
for the keton reduction by HLADH/NADH and the subsequent
recyclization of NAD+ by dithionite.
@ Assign the pro-R hydrogen and the pro-S hydrogen of NADH.
@ Compare typical applications of NAD+/NADH und FAD as
oxidoreductases with regard to their involvements in the biochemical
processes.
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Problems - 3
@ Nature exploits the reactivity of C-8 methyl group of flavins in order to
link to the apoenzyme via a SH group of a cystein. The overall process
is as given below. The last step is the oxidation of the cystein bound
FADH2 to enzyme-FAD.
Me
Me
R
N
O
N
N
N
H
CyS-
CySCH2
Me
R
N
O
N
N
N
H
O
O
@ Discuus the similarity of decarboxylation of a β-keto acid and
enzymatic pyruvate decarboxylation.
@ The pyrimidine-amino-group in thiamin pyrophosphate contributes in
the enzymatic TPP-dependent processes. How can this be explained?
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Enzymatic reactions and catalysis
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Vitamins, coenzymes, cofactors, enzymes
protein + coenzyme (vitamin)
enzyme
protein + cofactor (metal ion)
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Vitamins, coenzymes, enzymes cofactors
Vitamins that are coenzyme
precursors
Ø D. Voet, J. G. Voet, Biochemistry, second edition ed., John Wiley &
Sons, New York, 1995.
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Structure of water soluble vitamins
NH2
+
S
HO
OH
OH
Me
N
Me
N
N
N
S S
niacin
nicotinic acid
CO2H
H
subunit of CoA-SH
H
H2N
N
N
N
vitamin C
ascorbic acid
NH
H
S
lipoate
pantothenate
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CO2H
O
N
OH
HN
H
n=3
Me Me O
OH
O
OH
n
HO
pyridoxine
vitamin B6
H
riboflavin
vitamin B2
N
CH3
O
N
O
O
HO
OH
HOCH2
OH
N HC
3
thiamine
vitamin B1
CH2OH
OH
H
O
O
H
CH2OH
OH
N
N
H3C
H
BO/BBM
H
CO2H
biotin
vitamin H
O
N
H
N
CO2H
N
H
vitamin B12
CO2H
folate
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BO/BBM/SKB
Vitamin B12
Ø Discovering Nature's Diverse Pathways to Vitamin B12: A 35-Year Odyssey: A. I.
Scott, J. Org. Chem. 2003, 68, 2529-2539..
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BO/BBM
Fat soluble vitamins
CH3
O
HO
CH3
H3C
CH3
O
n
CH3
O
vitamin E
α-tocopherol
n=3
vitamin K1
Me
Me
CH2OH
Me
Me
vitamin A (all-trans)
H
HO
vitamin D3
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Vitamin C
• mp 190-192°C
• pK1 = 4.17; pK2 = 11.57
• anti-scorbutically factor (essential for the synthesis of
collagen)
• reducing agent in hydroxylation reactions
• radical scavenger (inhibitor)
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Industrial synthesis of vitamin C (Reichstein, 1934)
Ø H. Beyer, W. Walter, Lehrbuch der Organischen Chemie, 1998.
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Enzymatic synthesis of vitamin C
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Vitamin D
• Metabolism of calcium and phosphorous
• Deficiency impairs bone formation (rickets)
• Excess causes calcification of soft tissues
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Photochemical formation of vitamin D3
C-25
C-1
∗ two step reaction
- first step: conrotatory photochemical ring opening
- second step: [1,7] sigmatropic antarafacial hydrogen shift (thermal)
∗ Woodward-Hoffmann rules
∗ Active form 1,25-dihydroxyvitamin D3
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Oxidoreduction: Pyridine nucleotide coenzymes
(NAD+/NADP+) (Vitamin B3, Niacin)
∗ NAD+/NADH; NADP+/NADPH
- Structure
- Oxidation reactions
-
Reduction reactions (Pyruvate → lactate; lactate
dehydrogenase)
Comparison with LiAlH4
Mechanism and stereochemistry (Prochirality)
H(R)
H3C
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H(S)
OH
H(R) and H(S) enantiotopic hydrogens
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Bioactive flavins
H
O
∗ vitamin B2 – riboflavin
∗ flavin mononucleotide
OH
HO
OH
(FMN)
∗ flavin adenine dinucleotide
N
Me
N
O
N
N
H
O
(FAD)
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Me
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Flavin Biochemistry
∗ Dehydrogenation
∗ Activation of oxygen, hydroxylation, oxygenation
∗ Electron transfer, electron transport, respiration chain,
etc.
∗ Light emission, bacterial luciferase
(bioluminescence)
∗ Photobiology: Photolyase, phototropism,
photomorphogenesis, circadian processes
(Cryptochrome, Phototropin, blue-light photo
receptors)
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Flavin structure
Ø F. Müller, Chemistry and Biochemistry of Flavoenzymes, Vol. I, CRC,
Boca Raton, 1991.
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Isoalloxazine
synthesis
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Redox processes of flavins (electron transfer/protonation)
R1
R1
R2
N
O
N
N
N
H
red
R1
R2
N
R1
N
O
Fl-red
Flox
O
N
N
H+
H
R1
R2
N
R1
N
H
N
O
N
H
O
O
FlHrad
red
R1
R1
R2
N
N
H
H
N
O
N
H+
H
O
FlH2
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R1
R2
N
R1
N
H
N
O
N
H
O
FlH-red
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Some enzymatic processes of flavin nucleotides
• dihydrolipoyl dehydrogenase
• L-amino acid oxidase
• succinate dehydrogenase
• NADH dehydrogenase
• luciferase catalyzed bioluminescence
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Enzymatic processes of flavin nucleotides
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8α
α-S-Cysteinylflavin: Binding of flavins at the protein
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FAD-dehydrogenase – oxidation of a dithiol
O
O
SH
+
O
dihydrolipoyl
dehydrogenase
FAD
O
S
SH
+
FADH2
S
H+
R
Me
N
Me
N
N
O
N
S
S
H
R
O
H+
S
S
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R
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Bioluminescence
•
•
•
•
•
•
•
•
•
•
•
•
Photoluminescence
Chemoluminescence
Bioluminescence
Triboluminescence
Electroluminescence
Lyoluminescence
Aquoluminescence
Sonoluminescence
Galvanoluminescence
…
…
XYZ-Luminescence
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Population of excited state by
some process XYZ
Electronically
excited state
hν
Ground state
hν = Ee − E g
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Bioluminescence
light
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Bacterial bioluminescence
AA
• enzyme catalyzed
reaction: luciferase
• reaction cascade
ultimately leads to the
formation of the
excited state (S1) of 4ahydroxyflavin
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LOV domain of Phototropin
• Genetic information
translated into protein
structure
• Enzymatic synthesis of
FMN
• Molecular self-assembly
• Input light, output
morphogenesis
∆ S. Crosson, K. Moffat, 2001.
∆ See also: http://www.uniregensburg.de/Fakultaeten/nat_Fak_III/GK/SP/download/Kottke
_Phototropins.pdf
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Pyruvate decarboxylase – cofactor thiamine
pyrophosphate (TPP)
O
O
O
H3C
H3C
O
NH2
H3C
CO2
H
+
N
N
H
+
S
N HC
3
O
O
P O
O
P O
O
O
thiamine pyrophosphate
TPP
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Pyruvate decarboxylase
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Benzoin-type condensation – compare its mechanism
to TPP catalyzed processes (transketolase)
H
2
R
R
H
O
H R
O
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O
CN
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Enzyme bound biotin
O
HN
H
N E
NH
O
S
E: enzyme
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Biotin – catalyzes carboxylation as a CO2 carrier
∗ carboxylation
of
activated
carbon
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Pyridoxal phosphate (PLP)
Enzyme
CH2OH
OH
HOCH2
N
CH3
O
O
P
O
O
H
O
OH
O
P
O
N
CH3
N
H
O
H
O
O
N
CH3
H
pyridoxine
vitamin B6
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pyridoxal phosphate
PLP
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enzyme covalently
bound to the
cofactor
37
PLP-catalyzed processes of amino acids in Nature
∗ transimination
Enzyme
∗ decarboxylation
∗ transamination
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H
O
O
N
∗ racemization
∗ Cα-Cβ bond cleavage
(„β-elimination“)
∗ α,β-elimination
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O
P
O
N
H
O
H
+
CH3
enzyme covalently
bound to the
PLP cofactor
38
Decarboxylation of an amino acid
∗ First step: Synthesis of amino acid-bound PLP from enzymebound PLP
∗ Second step: Decarboxylation
∗ Third step: Transimination leading to enzyme-bound PLP
and „free“ decarboxylated amino acid
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Vitamin B12
∆ Discovering Nature's Diverse Pathways to Vitamin B12:
A 35-Year Odyssey, A. I. Scott, J. Org. Chem. 2003, 68,
2529-2539.
∆ Total synthesis of cobyric acid: historical development
and recent synthetic innovations, D. Riether, J. Mulzer,
European Journal of Organic Chemistry 2003, 30-45.
∆ W. Kaim, B. Schwederski, Bioanorganische Chemie,
Teubner, Stuttgart, 1991.
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Coenzym B12
• vitamin B12 coenzyme
(adenosylcobalamin)
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General scheme for the structure interconversion
cataylyzed by B12
C1
C2
X
H
Y
C1
C2
H
X
Y
1,2 shift process
• radical mechanism
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Processes requiring coenzym B12 - 1
glutamate
mutase
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Processes requiring coenzym B12 - 2
Ø H. Dugas, Bioorganic Chemistry - A Chemical Approach to Enzyme Action, 3rd
ed., Springer Verlag, New York, 1996, chapter 7.
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