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control of metabolic reactions. making +ve D G o ‘ reactions happen points of control: D G o ‘ and equilibrium multi-active enzymes: enzyme complexes and multiple active sites. reactions with +ve D G o ‘ can occur by:. coupling with a reaction with –ve D G o ‘ - PowerPoint PPT Presentation
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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!)