control of metabolic reactions•making +ve Go‘ reactions happen

•points of control:Go‘ and equilibrium •multi-active enzymes: enzyme complexes

and multiple active sites

reactions with +ve Go‘ can occur by:

• coupling with a reaction with –ve Go‘ • –ve physiological G due to cellular low

ratio [products]/[reactants]

1. reactions with +ve Go‘ occur by coupling with a reaction with -ve Go‘

• Thus, ATP ADP +Pi (G<0) is coupled with non-spontaneous reactions (G>0)

glucose-6-P + ADP

hexokinase

Glucose glucose-6-P + H20

G = 13.8 kJ.mol-1

ATP +H20 ADP +Pi

G = -30.5 kJ.mol-1

Glucose + ATP overall

G = -16.3 kJ.mol-1

2. Recall:

Go' of a reaction may be positive, and G negative, depending on cellular concentrations of reactants and products.

For a reaction A + B C + D

G = Gº' + RT ln[A] [B][C] [D]

any [products] or [substrate] that moves the reaction away from equilibrium ratio causes reaction to proceed spontaneously forward to restore equilibrium

Many reactions for which Go' is positive are spontaneous in vivo because other reactions cause [products] or [substrate].

At equilibrium, no net change so G = 0.

G = Gº' + RT ln

= Gº' + RT ln

Gº' = - RTln

defining K'eq =

Gº' = - RT ln K'eq

[C] [D][A] [B]

[C] [D][A] [B]

[C] [D][A] [B]

[C] [D][A] [B]

free energy change is related to the equilibrium constant (K'eq) = the ratio of

[products]/[reactants] at equilibrium

I won’t be asking you to solve any of these equations!

many reactions are near equilibrium

• then G ~0 (no net change in free energy)• easily reversed by changing ratio of

[products]/[substrate] as don’t need to overcome high G

For A+B ↔C+D product A+B C+D substrate A+B C+D

• enzymes that catalyse such reactions act to restore equilibrium

• rate regulated by [products]/[reactants]

Implication:

a reaction near equilibrium may have +ve Go' but be spontaneous in the cell

ve G because other reactions cause [products] or [substrate].

Other reactions are FAR from equilibrium

• enzyme rate is too slow to allow products to build to equilibrium concentration

• [substrate] builds up in excess of Keq

G <<<0 (highly negative)• not affected by [substrate] (saturated)• essentially irreversible• rate controlled by changing activity of

enzyme (eg allosteric interactions)

• reactions with G <<0 are often sites of regulation

1. Often occur early as a “committed step” in metabolic pathways (eg AcetylCoA carboxylase)

2. most metabolic pathways are irreversible • ≥ 1 step with -ve G required to drive: eg PDH,

pyruvate carboxylase)• one way street: return by a different street3. catabolic and anabolic pathways are separate

independent control (eg glycolysis and gluconeogenesis (eg pyruvate carboxylase) use different enzymes)

reactions with G <<0 are often sites of regulation

Pyruvate dehydrogenase

a pretty, pink multi-enzyme complex

‘gatekeeper’ to entry to citric acid cycle

http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/pdc/pdc.html

pyruvate dehydrogenase

6 NADH

2 NADH

2 NADH

Pyruvate dehydrogenase

regulates entry into the citric acid cycle of metabolites leaving glycolysis

Summary

1. structure of PDH complex3 enzymes (E1, E2, E3)

2. reactions of PDH complex5 reactions, 5 cofactors

3. mechanism of PDH complexlipoamide swinging arm

4. regulation of PDH complexde/phosphorylation of E1product inhibition of E2 and E3

Excellent animation of PDH reactions if you can access it: (not examinable, but might help understanding!)

http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/pdc/pdc.html

3 different ENZYMES 5 COFACTORS(non-covalently associated)

E1: pyruvate dehydrogenase +TPP

E2: dihydrolipoyl transacetylase + lipoamide

E3: dihydrolipoyl dehydrogenase + FAD

+ NADH+CoenzymeA

PDH = multi-enzyme complex

5 sequential reactions

catalyse

overall…..

pyruvate AcCoA

NAD+

NADH

CoA

CoA

CO2

high energy bond

irreversible(3C)

(2C)

multi-enzyme complex (E. coli)• a) dihydrolipoyl transacetylase (E2)

arranged as corners of a core cube

surrounded by an outer cube:

• b) pyruvate dehydrogenase (E1) edges

• c) dihydrolipoyl dehydrogenase (E3) facesNote that there are many copies of each enzyme in each complex

PDH structure is more complex in other organisms

dodecahedron core=• 12 pentagon faces

• 20 vertices (E2 trimers)

• in mammals =

+ kinase

+ phosphatase

E2 core of B. stearothermophilus

each enzyme uses a cofactor

2 lipoate binding domains in each E2

FAD in each E3

TPP in each E1

5 sequential reactions

21

3

5

4

pyruvate dehydrogenase

(E1)

dihydrolipoyl transacetylase

(E2)

dihydrolipoyldehydrogenase

(E3)

In summary: 1) pyruvate is decarboxylated hydroxyethyl, requires TPP to stabilise the intermediate. 2) hydroxyethyl oxidised to acetyl, collected by lipoamide of E2, which gets reduced. 3) lipoamide of E2, passes acetyl to coenzyme A acetyl CoA. 4) lipoamide of E2, gets re-oxidised, gives its electrons to FAD in E3 which 5) passes electrons to NAD NADH

1. decarboxylation by E1

Pyruvate

hydroxyethyl-

loss of CO2 conversion of pyruvate to a 2 carbon moiety

E1 has a bound coenzyme (TPP) that attacks pyruvate and stabilises the intermediate(3C)

(2C)

i. TPP forms a carbanion

H+ readily dissociates (due to adjacent N+)

N+ stabilises the carbanion

H+

releases CO2

CO2

ii. nucleophilic attack by TPP carbanion on electron-deficient C2 of pyruvate

hydroxyethyl-TTP

iii. TTP stabilises the carbanion intermediate after CO2 is lost.

CO2

can’t just remove CO2 highly unstable intermediate

I won’t ask you to recreate bond rearrangements!

- R - lys

2. formation of acetyl by E1

REDUCTIONgain of hydrogen

dihydro-lipoamide

OXIDATION

hydroxyethylacetyl-

lipoamide

- R - lysine

+ TTP regenerated

E2

hydroxyethyl is transferred the lipoamide group of E2, Lipoamide (= lipoic acid linked covalently to Lysine) contains a cyclic disulfide reactive group that can be reversibly reduced dihydro-lipoamide

E2 uses lipoamide as a cofactor

lipoic acid acts as a long flexible arm that can transfer substrates between active sites

there are actually 2 lipoate-binding domains in each E2.

cyclic disulphide

reversibly reduced and oxidised

lipoamide

= lipoic acid covalently bound to lysine in E2

3. trans-esterification

acetyl group transferred by E2 to CoA

= high energy thioester bond

• Form between carboxylic acid (COOH) and a thiol (SH) eg thiol in CoenzymeA

• eg Acetyl-CoA is common to CHO, fat and protein metabolism

• eg.In citric acid cycle, cleavage of thioester in succinyl-CoA provides energy for synthesis of GTP

Thioesters: high energy bond

Lipoamide cofactor in E2

• So… lipoamide swings to E3 to be reoxidised and transfer electrons to NADH via FAD

now we have acetyl-CoA

Kreb’s, FA synthesisnext must regenerate lipoamide and produce NADH

Remember: there are multiple copies of each enzyme in complex

So far….• disulfide swings to outer

shell to collect hydroxyethyl from TPP in E1

• swings to E2 to transfer acetyl to CoA

REDUCTIONin E3

OXIDATIONin E2

4. regeneration of lipoamide (E2) by FAD (E3)

REDUCTIONin E2

OXIDATIONin E3

NAD+ NADH + H+

• FAD funnels electrons to NAD+ NADH

• regeneration of FAD in E3

5. redox

ENZYME COFACTORE1: pyruvate dehydrogenase +TPPE2: dihydrolipoyl transacetylase + lipoamideE3: dihydrolipoyl dehydrogenase + FAD

PDH controlled by covalent modification and product inhibition

• mammalian complex also contains kinase and phosphatase

active E1

inactive E1

PDHkinase

P

PDHphosphatase

Ser

pyruvate AcCoA

NAD+ NADH

CO2

ATP

inhibit PDH• high energy state

active E1

inactive E1

PDHkinase

P

PDHphosphatase

Ser

pyruvate AcCoA

NAD+ NADH

CO2

activates

ATP

inhibition by products

in addition to activating PDH kinase,

NADH and acetyl-CoA:

• compete with substrates for binding sites

• drive E2 and E3 in reverse (these reactions are close to equilibrium)

• E2 not available to collect hydyrxyol from TPP

• TPP cannot accept pyruvate

activate PDH• low cell energy, or high available fuel

active E1

inactive E1

PDHkinase

P

PDHphosphatase

Ser

pyruvate AcCoA

NAD+ NADH

CO2

glucose

Insulin

activates

ADP

CoA

activate PDHpyruvate overrides NADH, AcCoA

still make AcCoA for fat when pyruvate

active E1

inactive E1

PDHkinase

P

PDHphosphatase

Ser

pyruvate AcCoA

NAD+ NADHCO2CoA

activates

pyruvate

AcCoA

glucose

citric acid cycle

OAA

malonyl-CoA

PDH

ACCarbox

gluc

oneo

gene

sis

fatty acids

Pyrcarbox

in high energy: (high ATP, high AcCoA, high NADH) gluconeogenesis, fatty acid synthesisin low energy (low ATP, low AcCoA, ) glycolysis

FASynthase

PEP

PK

CO2

CO2

CO2

We now look at 3 other enzymes that use ‘swinging arm’ cofactorsPyruvate carboxylaseAcetylCoA carboxylaseFatty acid synthase

pyruvate carboxylase

• first reaction in gluconeogenesis • with PEPCK to bypass pyruvate kinase

(G<<0 in glycolysis)• requires ATP to overcome –ve Go‘ of

glycolysis

+ HCO3-pyruvate

(3C)

oxaloacetate

(4C)

ATP ADP

glucose

(6C)

another good animation, if you can access it: (not examinable, but might help understanding!)

http://www.bmb.uga.edu/8010/moremen/weblinks/nucleotide/PyrCarb/PyrCarb.html

•tetramer•each monomer has 2 active sites•uses biotin as swinging arm

pyruvate carboxylase

biotin

HCO3-

biotin’s swinging

arm

carboxyphosphate

ATP

ADP

carboxybiotin

in active site 1

Biotin carboxylation is catalyzed at one active site : first, ATP reacts with HCO3-(bicarbonate) to yield carboxyphosphate. The carboxyl from this high energy phosphate intermediate is transferred to the nucleophilic N of the biotin ring

At active site 1:1. bicarb + ATP

high energy carboxyphosphate intermediate

2. -ve G transfer of CO2

to biotin = carboxylation

I won’t ask you to recreate bond rearrangements!

biotin

HCO3-

pyruvate(3C)

oxaloacetate

(4C)

carboxyphosphate

ATP

ADP

carboxybiotin

2. biotin arm swings to the 2nd active site,

active CO2 is transferred from carboxybiotin to pyruvate OAA

at active site 2:

1. CO2 leaves biotin, 2. biotin accepts a proton from pyruvate

3. pyruvate attacks CO2

OAA

nucleophile (donates e-)

I won’t ask you to recreate bond rearrangements!

pyruvate loses a proton, becomes an enolate

biotin

HCO3-

biotin’s swinging

arm

pyruvate(3C)

oxaloacetate

(4C)

carboxyphosphate

ATP

ADP

carboxybiotin

Overall:at active site 1: biotin + ATP + HCO3- carboxybiotin + ADP + Piat active site 2: carboxybiotin + pyruvate OAA + biotin

AcetylCoA carboxylase

• first reaction committed step in fatty acid synthesis

•Also uses biotin as swinging arm between two active sites•reactions very similar to pyruvate carboxylase

+ HCO3-AcetylCoA

(2C)

malonylCoA

(4C)

ATP ADP

fatty acids

biotin

HCO3-

biotin’s swinging

arm

Acetyl-CoA(2C)

malony-lCoA

(3C)

carboxyphosphate

ATP

ADP

carboxybiotin

WOW look! mechanism of carboxylation (addition of COO-) is the same as for pyruvate carboxylase!!! ATP-dependent carboxylation of the biotin, carried out at active site 1 , is followed by transfer of the carboxyl group to acetyl-CoA at a second active site 2 . only difference is COO- is added to acetylCoA rather than to pyruvate

regulation of AcCoA-Carboxylase

The mammalian enzyme is regulated, by phosphorylation by cAMP dependent kinase

inhibition when energy (cAMP)

allosteric control by local metabolites.

Conformational changes with regulation: active = multimeric filamentous complexes. inactive = dissociation to = monomeric form

P

fatty acid synthase• dimer• 6 active sites are individual domains of a large

protein – ? developed from gene fusion– has more catalytic activities than any enzyme!

• has two prosthetic groups thioester bonds– thiol of cysteine (in condensing domain)– thiol of P-pantetheine (in acyl carrier domain)

• acts as a long flexible arm transferring substrates between active sites

has two prosthetic groups

OPOH2C

O

OC

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

CH2 CH

NH

C O

-mercaptoethylamine

pantothenate

serine residue

phosphopantetheine of acyl carrier protein

phosphate

Phosphopantetheine is covalently linked to a serine of the acyl carrier protein domain  

The long flexible arm of phosphopantetheine allows its thiol to move between active sites

forms thioesters like CoA does

H3N+ C COO

CH2

SH

H

cysteine

thiol of cysteine in condensing domain

thiol of P-pantetheine

phosphopantetheine is part of CoA

N

N N

N

NH2

O

OHO

HH

H

CH2

H

OPOPOH2C

O

O O

O

P

O

O O

C

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

-mercaptoethylamine

pantothenate

ADP-3'- phosphate

Coenzyme A

phosphopantetheine

OPOH2C

O

OC

C

C

NH

CH2

CH2

C

NH

CH3H3C

HHO

O

CH2

CH2

SH

O

CH2 CH

NH

C O

-mercaptoethylamine

pantothenate

serine residue

phosphopantetheine of acyl carrier protein

phosphate

fatty acid synthase

2)Thioester bond between malonyl and pantetheine 3)The condensation reaction * involves decarboxylation of the malonyl carbanion attacks carbonyl carbon of the acetyl. Uses swinging arm of pantotheineYou will have done these reactions in Dr Denyer’s lectures

2NADPH H20

32

dimer of the multi-domain enzyme are probably aligned in antiparallel

Pant-SH HS-Cys

Cys-SH HS-Pant

Fatty Acid Synthase dimer

In the transfer step:the growing fatty acid chain is preferentially passed from the pantetheine thiol of one subunit cysteine thiol of the other

? intra-subunit substrate transfers also occur by swinging arm of pantetheine

essential dietary cofactors: cannot be made by mammals

• thiamine = vitamin B1 (in TPP) – deficiency = beri-beri– eg alcohol reduced uptakeof thiamine

brain symptoms (brain glucose metabolism)

• riboflavin = vitamin B2 (for FAD)• niacin = vitamin B3 (NAD)• lipoic acid• biotin• pantothenic acid (vitamin B5)

advantages of multi-active site enzymes and multi enzyme complexes

diffusion distance between substrate and active sites (usually the limiting factor in determining the reaction rate)

reaction ratechance of side reactions

– substrates stay within complex

• coordinated control of sequential reactions

Voet, Voet and Pratt (2nd Ed)

• .G and equilibrium pg 401

• PDH pg 519 -524, regulation pg 533

• TPP mechanism pg 450

• thioester bonds pg 413

• Pyruvate carboxylase pg 502, pg

• AcetylCoA carboxylase pg 651• Fatty acid synthase pg 653 (much more detail than

you need for this lecture!)