Transcript Pyruvate Dehydrogenase

Metabolism – Intro to
Metabolism
CH339K
Going back to the early lectures
G  H  TS
S  K ln W
 G   RT ln K eq
0
 G
K eq  e
o
RT
 G   G  RT ln
0
[ Products ]
[Reactants ]
Why the big Go’ for Hydrolyzing
Phosphoanhydrides?
• Electrostatic repulsion
betwixt negative
charges
• Resonance stabilization
of products
• pH effects
pH Effects – Go vs. Go’
 G   G  RT ln
o
 G   G  RT ln
o
 G   G  RT ln
o
   10
At pH 7, H

 Products 
 Reactants 
 ADP Pi H  
 ATP H 2 O 
 ADP Pi 
 RT
 ATP 
-7
 G '   G  RT ln
o
o
 G '   G  RT ln
o
o
H 

ln
H 2 O 
M
 ADP Pi 
7
 RT ln 10 
 ATP 
 ADP Pi 
kJ
 41 . 5
 ATP 
mol
G in kcal/mol)
WOW!
Cellular Gs are not Go’ s
Go’ for hydrolysis of ATP is about -31 kJ/mol
Cellular conditions are not standard, however:
In a human erythrocyte,
[ATP]≈2.25 mM, [ADP] ≈0.25 mM, [PO4] ≈1.65 mM
 G Hyd   G ' RT ln
o
 G Hyd   31
 G Hyd   31
kJ
[ ADP ][ Pi ]
[ ATP ]
 8 . 315
mol
kJ
mol
 (  21
J
K  mol
kJ
mol
 298 K  ln
)   52
(. 00025 M )(. 00165 M )
(. 00225 M )
kJ
mol
Unfavorable Reactions can be
Subsidized with Favorable Ones
Activation with ATP - luciferin
Excited state
of oxyluciferin
forms and
decays
For those who prefer more detail
Excerpted from Baldwin, T. (1996) Structure 4: 223 – 228,
Just because it’s cool…
Tobacco seedling w/ cloned
luciferase
Southeast Asian firefly tree
Just because it’s cool…
Firefly squid (Watasenia
scintillans ) of Toyama Bay,
Japan
New Zealand glowworm
(Arachnocampa) cave
Hydrolysis of Thioesters can also provide a lot of
free energy
Acetyl Coenzyme A
Sample Go’Hydrolysis
“Phosphate Transfer Potential”
is a fancy-schmancy term for –Go’
Electrochemistry in
review
One beaker w/ ZnSO4
and a Zn electrode
One beaker w/ CuSO4
and a Cu electrode
Zinc gets oxidized and
the electrode slowly
vanishes
Copper gets reduced
and the electrode gets
fatter
1.10 V
Standard Hydrogen Electrode
Redox Table
• Higher the SRP, the
better the oxidant
• Lower the SRP, the
better the reductant
• Any substance can
oxidize any substance
below it in the table.
• The number of
reactants involved
doesn’t change the
reduction potential
• i.e. if a reaction involves
2 NAD+, the SRP is still
-0.32 V
Electrochemistry in
review
1.10 V
Zinc gets oxidized
Copper gets reduced
What determines who
gets oxidized?
 E total  E acceptor - E donor
o
o
o
Eo and Keq
 E total  E acceptor - E donor
o
o
o
For an actual half reaction aA + ne- ⇌ aAE E 
o
RT
(Analagous to the relation between G and Go’)
- a
ln
[A ]
nF
[A]
a
For an actual redox reaction:
A+n + ne- ⇌ A
B
⇌ B+n + neA+n + B
⇌ A + B+n
Ea  E 
o
a
RT
nF
ln
[A]
[A
n
and
]
Eb  E
o
b

RT
nF
ln
[B]
[B
n
]
Eo and Keq (cont.)
At equilibrium, the two are equal:
RT
Ea 
o
Combining:
ln
nF
E E
o
a
o
b
[A]
[A

n
RT
 Eb 
o
]
[A]
ln
nF
[A
n
RT
nF

]
[B]
ln
[B
RT
]
[B]
ln
nF
n
[B
n
]
Or
E
o
 E E
o
a
o
b

RT
nF
ln
[A][B
[A
n
n
]
][B]
Or
E
o
 Ea  Eb 
o
o
RT
lnKeq
nF
Or (rearranging)
nF
Keq  e
RT
ΔE
o
Dr. Ready gets to the Point!
Eo and Go
So:
nF
K eq  e RT
But we already know:
K eq  e
ΔE
 ΔG
o
o
Another Point!
RT
Therefore:
 G   nF  E
o
o
NAD+ Reduction
(Nicotinamide Adenine Dinucleotide)
NAD+ is a common redox cofactor in biochemistry
Coenzyme Q
Coenzyme Q is another electron carrier in the cell
An Example:
What is Go’ for the
Oxidation of NADH by
Ubiquinone?
Cigarettes ≠ Vitamins
“Organic” ≠ “Healthy”
LD50
0.5 – 1.0 mg / kg
Vomiting and nausea, diarrhea, Headaches, Difficulty breathing,
Pallor, Sweating, Palpitations, Lisps, Stomach pains/cramps,
Seizures, Weakness, Drooling, and - of course - Death
Flavins
•Energy (ATP)
Catabolism
(Oxidation)
•Parts (amino acids, etc.)
Anabolism
•Reducing Power (NADH, NADPH) (Reduction)
Fates of Glucose
Catabolism of Glucose
C6H12O6 + 6O2 → 6CO2 + 6H2O
Go’ = -2870 kJ/mol
It takes 31 kJ/mol to make an ATP. Enough
energy is available for making ~90 (theoretically)
An aside on diets
Glucose (a carb), mol. wt. = 180 g/mol
-2870 kJ/mol = -686 kcal/mol
-686 kcal/mol / 180 g/mol = 3.8 kcal/g
Palmitic Acid (a fatty acid) mol. wt. = 256
g/mol
-9959 kJ/mol = -2380 kcal/mol
-2380 kcal/mol / 256 g/mol = 9.3 kcal/g
Alanine (an amino acid) mol. wt. = 88 g/mol
-1297 kJ/mol = -310 kcal/mol
-310 kcal/mol / 88 g/mol = 3.5 kcal/g
An aside on diets (cont.)
From Nutristrategy.com:
Fat: 1 gram = 9 calories
Protein: 1 gram = 4 calories
Carbohydrates: 1 gram = 4 calories
The diet values come from the Go’ for
oxidizing the various biomolecules.
Catabolism of Glucose
Interconversion of C6 Sugars
Glycogen
Glucose-1-Phosphate
-7.3 kJ/mol
Glucose
Glucose-6-Phosphate
Amino Sugars
-0.4 kJ/mol
Fructose-6-Phosphate
Nucleotides
Fatty Acids
Catabolism
STOP HERE FOR INTRO LECTURE
Glucose Catabolism Part 1:
Glycolysis
•
•
•
•
•
Aka Embden-Meyerhof pathway
Worked out in the 1930’s
Partially oxidizes glucose
Uses no O2
Takes place in cytoplasm
Interconversion of C6 Sugars (Again)
Glycogen
Glucose-1-Phosphate
Phosphoglucomutase
Glucose
-7.3 kJ/mol
Glucose-6-Phosphate
Phosphohexose isomerase
Amino Sugars
-0.4 kJ/mol
Fructose-6-Phosphate
Nucleotides
Fatty Acids
Catabolism
Don’t Eat the Toothpaste!
• Phosphoglucomutase
contains a PO4-2 group
attached to residue D8.
• Fluoride has a number
of toxic effects
• One of them is the
removal of the phosphate
from phosphoglucomutase
• No phosphate = no
activity
• No activity = can’t utilize
glycogen
Glycolysis - Energetics
Phosphohexose Isomerase
Aldolase
Aldolase Reaction
• The standard free energy , Go,for the aldolase
reaction is very unfavorable (~ +25 kJ/mol)
• Under cellular conditions, the real free energy, G, is
favorable (~ -6 kJ/mol)
• [G-3P] is maintained well below the equilibrium level by
being processed through the glycolytic pathway
Triose Phosphate Isomerase
Gyceraldehyde-3-P Dehydrogenase
Phosphoglyceromutase
H8 in human
erythrocyte PGM
Overall Reaction
The overall reaction of glycolysis is:
Glucose + 2 NAD+ + 2 ADP + 2 Pi
2 pyruvate + 2 NADH + 2 ATP + 2 H2O + 4 H+
• There is a net gain of 2 ATP per glucose
molecule
• As glucose is oxidized, two NAD+ are reduced
to 2 NADH
When two things look alike…
…there can be a problem.
Arsenate Poisoning (in part)
• G3P Dehydrogenase will happily use arsenate as a
substrate.
• 1-Arseno-3-phosphoglycerate decomposes
spontaneously without production of ATP.
• Primary poisoning effect is on a different part of
catabolism
Why does arsenic poisoning ever come up?
•
•
•
•
•
•
•
Chromated copper arsenate was the primary agent for
pressure treated wood in the USA until 2003
Mono- and disodium methyl arsenate are used as
agricultural insecticides
Arsphenamine was one of the first treatments for syphilis
Arsenic trioxide is an approved treatment for
promyelocytic leukemia
Lewisite is an old-fashioned CBW blister and lung agent
Coppers acetoarsenite is “Paris green,” a pigment used
by artists, some of whom had the habit of licking their
brushes
Scheele’s Green (copper arsenite) was used as a
coloring agent for candy in the 19th century
Relation to Hb Oxygenation
Glycolysis – Genetic Defects
Antitrypanosomals
Remember these guys?
• Chagas Disease
• African Sleeping Sickness
• Nagana
• Leishmaniasis (“Baghdad
Boil”)
• Afflict hundreds of millions
• Nagana responsible for the
popularity of cannibalism in the
African “fly belt.”
• Leishmaniasis is now endemic
in Texas
Antitrypanosomals
• Trypanosomes have unusual glycolysis
enzymes
• First 7 steps carried out in “glycosomes”
• Enzymes are quite different in structure and
sequence from mammalian enzymes
• Good drug targets
Antitrypanosomals
Model of L. mexicana glyceraldehyde-3-phosphate dehydrogenase complexed with N6-(1naphthylmethyl)-2¢-deoxy-2¢- (3-methoxybenzamido)-adenosine.
Antitrypanosomals
Binding mode of 2-amino-N6-(p-hydroxyphenethyl)adenosine to T. brucei
phosphoglycerate kinase.
Energetics of Glycolysis
Go values are scattered: + and G in cells is revealing:
• Most values near zero
• 3 of 10 Rxns have large, negative G (i.e. irreversible)
• Large negative G Rxns are sites of regulation!
Glycolysis - Regulation
Hexokinase regulation
• Hexokinase – muscle
– Km for glucose is 0.1 mM; cell has 4 mm glucose
– So hexokinase is normally active!
– Allosterically inhibited by (product) glucose-6-P
(product inhibition)
• Glucokinase – liver, pancreas
Km glucose ≈ 8 mM (144 mg/dl – above normal)
Cooperative – nH ≈ 1.7
No product inhibition
Only turns on when cell is rich in glucose
Shifts hepatocytes from “fasting” to “fed” metabolic
states, encouraging glycogen synthesis and glycolysis
– Acts as signal in pancreas to release insulin
–
–
–
–
–
Hexokinase vs. Glucokinase
PFK
•
•
•
PFK is a tetrameric protein that exists in two conformational states - R
and T (i.e. cooperative)
High concentrations of ATP shift the T⇄R equilibrium in favor of the T
state decreasing PFK’s affinity for F6P
AMP, ADP and Fructose 2,6 Bisphosphate acts to relieve inhibition by
ATP
Fates of Pyruvate
Pyruvate
Ethanol
(Yeast, no O2)
Lactate
(Critters, no O2)
AcetylCoA
(Aerobic)
In the absence of O2, no further oxidation
occurs. NADH builds up, and NAD+ has to
be regenerated to continue glycolysis
NADH Regeneration
Yeasties: Alcohol Dehydrogenase
Alcohol
Dehydrogenase
Pyruvate
Decarboxylase
Critters: Lactate Dehydrogenase
Lactate
Dehydrogenase
Glucose Catabolism Part 2
Pyruvate Dehydrogenase
• Huge multienzyme complex
– 4.6 Mdaltons in E. Coli (a24b24g12)
– 9 Mdaltons in mammals (a60b60g24)
• 3 separate enzyme functions create overall
reaction
Pyruvate + NAD+ + HSCoA  CO2 + Acetyl CoA + NADH
• This is where we actually lose our first
carbon(s) from glucose
Pyruvate Dehydrogenase - Reaction
PDH - Subunits
Subunit
Enzyme Function
Cofactor
Number
In
Prokaryotes
Number
In
Eukaryotes
a (or E1)
Pyruvate
Dehydrogenase
Thiamine
24
Pyrophosphate
30
b (or E2)
Dihydrolipoamide
Transacetylase
Lipoic Acid
24
60
g (or E3)
Dihydrolipoamide
Dehydrogenase
Flavin Adenine
Dinucleotide
12
12
PDH - Structure
PDH - Schematic
E1 – Pyruvate Dehydrogenase Proper
•
•
•
•
In E. coli, E1 is a dimer of two similar subunits
In mammals, E1 is an a2b2 tetramer.
Each E1 contains 2 active sites
Each active site contains a thiamine
pyrophosphate cofactor.
• TPP is ligated to a metal ion and is H-bonded
to several amino acids
Pyruvate Dehydrogenase – Thiamine
Pyrophosphate
Hydrogen is
Acidic
Pyruvate Dehydrogenase
E2 – Dihydrolipoamide Transacetylase
Lipoic Acid
• In enzyme, Lipoic Acid
is attached to a lysine
• Disulfide is at end of
very long floppy arm
• Can bounce back and
forth between PDC and
DHLD on surface
S
S
C H2
C H2
C H2
C H2
COOH
Coenzyme A
• Thioesters are activated compounds
• Coenzyme A is a common activator
• Warhead of CoA is the thiol
– Hence, abbreviated HS-CoA
Dihydrolipoamide Transacetylase
H2
C C C C COOH
H2 H2 H2
O
H3C
O
T hia min e P yr o p h os p h at e
+
H3C
S
S
H2
C C C C COOH
H2 H2 H2
S
H2
C C C C COOH
H2 H2 H2
S
+
HS
S
O
H3C
H
S
H
H2
C C C C COOH
H2 H2 H2
S
S
H
H
+
O
+
T hia min e P yr o p h os p h at e
C o e nz y m e A
H3C
S
C o e nzy m e A
• Lipoamide is reduced
• Accepts acyl unit from PDC / Thiamine PP
• Transfers to CoA
FAD
E3 - Dihydrolipoamide Dehydrogenase
H2
C C C C COOH
H2 H2 H2
S
S
H
H
+
FAD
H2
C C C C COOH
H2 H2 H2
S
S
+
F A D H2
PDH - Overall
H 2O
HS
R'
As
O
S
R'
+
HS
As
S
R
R
Organic arsenicals are potent inhibitors of lipoamidecontaining enzymes such as Pyruvate Dehydrogenase.
These highly toxic compounds react with “vicinal” dithiols
such as the functional group of lipoamide.
PDH Regulation
Product inhibition by NADH & acetyl CoA:
 NADH competes with NAD+ for binding to E3.
 Acetyl CoA competes with CoA for binding to E2.
PDH - Regulation
Regulation by E1 phosphorylation/dephosphorylation:
Specific regulatory Kinases & Phosphatases associated with
Pyruvate Dehydrogenase in the mitochondrial matrix:
 Pyruvate Dehydrogenase Kinases catalyze
phosphorylation of serine residues of E1, inhibiting
the complex.
 Pyruvate Dehydrogenase Phosphatases reverse this
inhibition.
Pyruvate Dehydrogenase Kinases are activated by NADH &
acetyl-CoA, providing another way the 2 major products of
Pyruvate Dehydrogenase reaction inhibit the complex.
During starvation:
 Pyruvate Dehydrogenase Kinase increases in
amount in most tissues, including skeletal muscle,
via increased gene transcription.
 Under the same conditions, the amount of Pyruvate
Dehydrogenase Phosphatase decreases.
The resulting inhibition of Pyruvate Dehydrogenase
prevents muscle and other tissues from catabolizing glucose
& gluconeogenesis precursors.
 Metabolism shifts toward fat utilization.
 Muscle protein breakdown to supply
gluconeogenesis precursors is minimized.
 Available glucose is spared for use by the brain.
THE KREBS CYCLE
Overall Reaction
Per glucose that entered glycolysis:
2AcCoA  4 CO
2
 2 ATP  6 NADH  2 FADH
Thus, at the end of the cycle, we will have
converted our glucose completely to CO2.
C 6 H 12 O 6  6 O 2  6 CO 2  6 H 2 O
We still won’t have used any oxygen or
made any water.
2
Location
• Also known as citric acid cycle, tricarboxylic acid cycle
• Krebs takes place in the mitochondrial matrix
• One enzyme is an integral membrane protein of the IMM
At Equilibrium
Citrate
91%
Cis-Aconitate
3%
Isocitrate
6%
Stereospecificity of Aconitase
• Recognized back in 1956 that aconitase dehydrates across a
particular bond in citrate (England et al (1957) J. Biol. Chem. 226: 1047)
• Citrate is not chiral
• Multipoint binding allows stereospecificity in a nonchiral compound
An Aconitase Inhibitor
•
•
•
•
Sodium Fluoroacetate is a fairly potent toxin (2-10 mg/kg)
Brand name 1080
Incoporated into fluoroacetylCoA, then into fluorocitrate
Fluorocitrate is a powerful competitive inhibitor of aconitase
Coyote Control by 1080
Isocitrate Dehydrogenase
Go’ = -20.9 kJ/mol
1) Oxidation: NAD+ oxidizes the hydroxyl carbon of
isocitrate
2) Decarboxylation: A Mn+2 bound to the enzyme
stabilizes the intermediate
3) Protonation: Reforms the carbonyl to generate
product
4) General Principle: NAD+ is usually the electron
recipient when oxidizing at a hydroxyl
•We’ve now lost 2 CO2 in Krebs + 1 in PDH – glucose is gone.
•The two carbons we’ve lost are not the same ones we
brought in.
•Substrate level phosphorylation
•Plants make ATP directly
•Critters make GTP, then exchange phosphate to ATP
Succinyl CoA Synthetase Rxn
1. CoA is displaced by an
Orthophosphate
2. The phosphate group is transferred
to a Histidine residue on the
enzyme
3. Succinate leaves as a product
4. The enzyme is dephosphorylated,
passing PO4-3 to a nucleotide
diphosphate
General Principle: FAD is the preferred cofactor for
oxidizing a carbon-carbon bond.
Succinate Dehydrogenase is an integral membrane
protein
Water attacks the double
bond in a 2-step process.
Go’
G
1.) Citrate Synthase
2.) Aconitase
3.) Isocitrate Dehydrogenase
4.) α-Ketoglutarate Dehydrogenase
5.) Succinyl-CoA Synthetase
6.) Succinate Dehydrogenase
7.) Fumarase
8.) Malate Dehydrogenase
9.) Overall reaction
Krebs Cycle Energetics
Reaction
1
2
3
4
5
6
7
8
Enzyme
Citrate synthase
Aconitase
Isocitrate dehydrogenase
a-Ketoglutarate dehydrogenase complex
Succinyl-CoA synthetase
Succinate dehydrogenase
Fumerase
Malate dehydrogenase
G°'
(kJ/mol)
-32.2
+6.3
-20.9
-33.5
-2.9
0.0
-3.8
+29.7
The citric acid is regulated
by three simple
mechanisms.
1. Substrate availability
2. Product inhibition
3. Competitive feedback
inhibition.
The Krebs cycle is amphibolic – intermediates are also used to make stuff.