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4/20/10
1
Cofactors
Manickam Sugumaran Department of Biology University of Massachusetts Boston, MA 02125
Holoenzyme can be dissociated into inactive apoenzyme and cofactor.
+
--->
Inactive Inactive Active
HOLOENZYME COFACTOR +
--->
APOENZYME
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2
Cofactors are derived from vitamins and other small molecules. Some times they are simply inorganic ions.
looselybound
tightlybound
Esssential ions inmetallo enzymes
CosubstratesLoosely bound
Prosthetic grouptightly bound
Coenzymes (organic)
Cofactors
Iron - sulfur clustrer found in some proteins
S
S
S
S
Fe
S Fe
SFe
SFe
S
[4Fe-S]
SSS
S S
SFeFe
[2Fe-S]
Fe - S cluster in aconitase
FeS
S
FeS
Fe
Fe SS
S
S
S
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3
Vitamins and coenzymes
• Vitamins are usually converted into coenzymes by simple biochemical reactions.
• One exception - Vitamin C is both the vitamin form and the coenzyme form.
Vitamin C - Ascorbic acid
O
O
OH
OH
HO
HOO
O
OH
OH
O
O
Ascorbic acid Dehydroascorbic acid
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4
Ascorbic acid is required for collagen proline hydroxylase reaction
Collagen Proline Hydroxylase reaction
HO OH
O
O
O
N
HH
O
O2+
α -ketoglutarate
Peptidylproline
Peptidyl4 - hydroxy proline
Succinate
N
H
O
OH
HOOH
O
O
Fe2+
(Ascorbic acid)
+ CO2
Lactate dehydrogenase follows sequential Bi Bi Mechanism
E E - NAD E - NADH
NAD Lactate Pyruvate NADH
EE NADLactate E
NADHPyruvate
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Lactate Dehydrogenase reaction
N
O
NH2R
O HH
H3COO
NN H
HN
HN NH2
N
O
NH2R
O
OO
H3C
NN
HN
HN NH2
H
H
H
H
Pro R and Pro S hydrogen and A and B side of NAD.
NR
O
NH2
Ha Hb
12
3
Ha Proton is Pro RHb Proton is Pro S
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Stereospecificity of NAD
CH3CD2OH + NAD -----> CH3CDO + NAD(D)
When labeled ethanol was used with NAD, one atom of deuterium was transferred to NAD and one atom was retained in acetaldehyde. None was lost to the solvent.
CH3CHO + NAD(D) -----> CH3CHDOH + NAD When labeled resultant NAD(D) was used to reduce acetaldehyde, all the deuterium was transferred from NAD(D) quantitatively to the ethanol that was formed. None was lost to solvent again.
Thus NAD stereospecifically transfers hydrogen atom to its substrates
Stereospecificity of Alcohol dehydrogenase reaction - ( A side specific dehydrogenase)
H3C OH
D D
NR
ONH2
HbD
H3C O
D
NR
ONH2
Ethanol Acetaldehyde
NAD NADH
One atom of D is specifically transferred to Pro R position (A side) of NAD by the enzyme. In the reverse direction, the Pro R hydrogen from NADH is specifically transferred to acetaldehyde to generate stereospecifically labeled ethanol.
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Pyridoxine is converted to pyridoxal phosphate, the coenzyme form of this vitamin.
Pyridoxal Phosphate
PyridoxineN
HOH2C OH
CH3
OH
HN
HOH2C OH
CH3
O
PyridoxalH
HN
OH
CH3
O
OPO
OO
PyridoxamineHN
HOH2C OH
CH3
NH2
Folate coenzymes
HN
N
N
N
O
H2N
Pterin
12
34 5
6
7
8
(2- amino -4-oxopteridine)
HN
N
N
N
O
H2N
N
O
NOH
O OH
O
H
H
Folate
NADPH
NADP
Dihydrofolate
HN
N
N
N
O
H2N
R
HH
H
NADPHNADP
HN
N
N
N
O
H2N
RH
HH
H
H
Tetrahydrofolate
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8
Biopterin and amino acid hydroxylases
NADPH
NADPDihydrofolate reductase
HN
N
N
N
O
H2N
CH3OH
OH
HHH
HH
TetrahydrobiopterinQuinonoid dihydrobiopterin
N
N
N
N
O
H2N
CH3OH
OH NADH NAD
Dihydropteridinereductase
COOHH2N
COOHH2N
HO
PhenylalanineTyrosineO2H2O
HN
N
N
N
O
H2N
CH3OH
OH
Biopterin Dihydrobiopterin
HN
N
N
N
O
H2N
CH3OH
OH
HHHNADPH NADP
Phenylalanine hydroxylase
Biotin - carboxylation reaction
B
O
OO
HAMP
O P O
OO P O
OO AMP
O P O
OO
Inversion at Phosphate
O
OOHPHO
OO
NHN
S
O
NHN
S
O
OO
O
H
PHO
OO
NHN
S
O
O
O
NHN
S
O
H
+
ATP Bicarbonate
CarboxyBiotin
ADP
Carboxyphosphate
OP
HOO
O
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9
Biotin - carboxylation reaction
NHN
S
O
O
O
O
O
H3CS
CoAO
H H
H3CS
CoAO
H
H
H3CS
CoAO
HOO
NHN
S
O
H3C
S CoA
OH
OO
H
NHN
S
OH
NHN
S
OH
Acetyl CoA
Malonyl CoABiotinylated Enzyme
NIH shift
DH
COOH
NH2
D
H
COOH
NH2
O
[O]
Phenylalanine Arene oxideOH
D
COOH
NH2
H
COOH
NH2
O
D
TyrosineDieneoneH
H
D
H
COOH
NH2
O
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10
Different states of flavins
12
3
4567
8
7a
8a 9 10 1a
4aN
N
NH
N
O
O
R
H3C
H3C N
N
NH
N
O
O
R
H3C
H3CH
H
+ 2H
- 2H
Oxidized flavin Reduced Flavin
N
N
NH
N
O
O
R
H3C
H3CH
N
N
NH
N
O
O
R
H3C
H3CH
Semiquinone radical
Covalent attachment of FAD to some enzymes
Enzyme bound FADH2
N
N
NH
N
O
O
R
H3C
H3C N
N
NH
N
O
O
R
H3C
H3CH
H
+ 2H
- 2H
Oxidized flavin Reduced Flavin
S
vinylogous α,β -unsaturated amine
N
N
NH
N
O
O
R
H3CH
H
S
N
N
NH
N
O
O
R
H3C
H
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11
FAD - two electron reduction
N
N
NH
N
O
O
R
H3C
H3C
Oxidized flavin Reduced Flavin
D2OH
NR
O
NH2
H
NR
O
NH2
NADNADH
+N
N
NH
N
O
O
R
H3C
H3C
H
D
+
From model compounds, the following generalized reduction scheme has been derived for FADH2 formation. The hydride is transferred to the N-5 and a proton from water is incorporated into N-1 during the reduction of FAD by NADH in water.
Glutathione reduction/oxidation
N
N
NH
N
O
O
R
H3C
H3C N
N
NH
N
O
O
R
H3C
H3CH
H
Oxidized glutathione
Reduced Flavin
H2NN
N
COOH
OSH
O
COOH
H
HH2N
NN
COOH
OS
O
COOH
H
H
NH2NN
COOH
OS
O
HOOC
H
H2
Oxidized flavin
Reduced glutathione
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12
Mechanism of thiol - disulfide exchange catalyzed by Flavin
N
N
NH
N
O
O
R
H3C
H3C
N
N
NH
N
O
O
R
H3C
H3CH
H
Reduced Flavin
Oxidized flavin SH
N
N
NH
N
O
O
R
H3C
H3C
SH
Thiol
S S
N
N
NH
N
O
O
R
H3C
H3C
SH
H
S
disulfide