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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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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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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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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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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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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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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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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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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