Metabolism – Intro to Metabolism CH339K. Going back to the early lectures.
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Transcript of Metabolism – Intro to Metabolism CH339K. Going back to the early lectures.
Metabolism – Intro to Metabolism
CH339K
Going back to the early lectures
][Reactants
]Products[ln
ln
ln
0
0
RTGG
eK
KRTG
WKS
STHG
RT
G
eq
eq
o
Why the big Go’ for Hydrolyzing Phosphoanhydrides?
• Electrostatic repulsion betwixt negative charges
• Resonance stabilization of products
• pH effects
pH Effects – Go vs. Go’
mol
kJ 5.41ln'
10lnln'
M10 7,pH At
lnln
ln
ln
7
7-
2
2
ReactantsProducts
ATP
PiADPRTGG
RTATP
PiADPRTGG
H
OH
HRT
ATP
PiADPRTGG
OHATP
HPiADPRTGG
RTGG
oo
oo
o
o
o
G in kcal/mol)
WOW!
Cellular Gs are not Go’ sGo’ 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
mol
kJ
mol
kJ
mol
kJG
M
MMK
molK
J
mol
kJG
ATP
PiADPRTGG
Hyd
Hyd
oHyd
52)21(31
)00225(.
)00165)(.00025(.ln298315.831
][
]][[ln'
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,
Tobacco seedling w/ cloned luciferase
Southeast Asian firefly tree
Just because it’s cool…
Just because it’s cool…
New Zealand glowworm (Arachnocampa) cave
Firefly squid (Watasenia
scintillans ) of Toyama Bay, Japan
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’
1.10 V
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
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
1.10 V
Electrochemistry in review
Zinc gets oxidizedCopper gets reduced
odonor
oacceptor
ototal E-EE
What determines who gets oxidized?
Eo and Keq
For an actual half reaction aA + ne- ⇌ aA-
For an actual redox reaction:A+n + ne- ⇌ AB ⇌ B+n + ne- A+n + B ⇌ A + B+n
and
odonor
oacceptor
ototal E-EE
a
a-o
[A]
][Aln
nF
RTEE
][A
[A]ln
nF
RTEE
noaa
][B
[B]ln
nF
RTEE
nobb
(Analagous to the relation between G and Go’)
Eo and Keq (cont.)
At equilibrium, the two are equal:
Combining:
Or
Or
Or (rearranging)
Dr. Ready gets to the Point!
][B
[B]ln
nF
RTE
][A
[A]ln
nF
RTE
nobn
oa
][B
[B]ln
nF
RT
][A
[A]ln
nF
RTEE
nnob
oa
][B][A
][A][Bln
nF
RTEEE
n
nob
oa
o
lnKeqnF
RTEEE o
boa
o
oΔERT
nF
eKeq
Eo and Go
So:
But we already know:
Therefore:
oΔERT
nF
eqK e
RT
ΔG
eq
o
K
e
oo EnFG
Another Point!
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 theOxidation 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)
•Parts (amino acids, etc.)
•Reducing Power (NADH, NADPH)Catabolism
(Oxidation)
Anabolism
(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
Glucose-1-Phosphate
Glucose-6-Phosphate
Fructose-6-Phosphate
Glycogen
Glucose Amino Sugars
Nucleotides
Fatty Acids
-7.3 kJ/mol
-0.4 kJ/mol
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)
Glucose-1-Phosphate
Glucose-6-Phosphate
Fructose-6-Phosphate
Glycogen
Glucose Amino Sugars
Nucleotides
Fatty Acids
-7.3 kJ/mol
-0.4 kJ/mol
Catabolism
Phosphoglucomutase
Phosphohexose isomerase
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
AntitrypanosomalsRemember 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-(1-naphthylmethyl)-2¢-deoxy-2¢- (3-methoxybenzamido)-adenosine.
Antitrypanosomals
Binding mode of 2-amino-N6-(p-hydroxyphenethyl)adenosine to T. brucei phosphoglycerate kinase.
Energetics of GlycolysisGo 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
AcetylCoAEthanol Lactate(Yeast, no O2) (Critters, no O2) (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
PyruvateDecarboxylase
AlcoholDehydrogenase
Critters: Lactate Dehydrogenase
LactateDehydrogenase
Glucose Catabolism Part 2Pyruvate Dehydrogenase
• Huge multienzyme complex– 4.6 Mdaltons in E. Coli (242412)
– 9 Mdaltons in mammals (606024)
• 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
(or E1) Pyruvate Dehydrogenase
Thiamine Pyrophosphate
24 30
(or E2) Dihydrolipoamide Transacetylase
Lipoic Acid 24 60
(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 22 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
Hydrogen is Acidic
Pyruvate Dehydrogenase
E2 – Dihydrolipoamide TransacetylaseLipoic 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
CH2
CH2
CH2
CH2
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
S S
CH2
CH2
CH2
CH2
COOH
CH3
O
H
SH Coenzyme A
S S
CH2
CH2
CH2
CH2
COOH
H H
H3C
O
S Coenzyme A
+
+
• Lipoamide is reduced• Accepts acyl unit from PDC / Thiamine PP• Transfers to CoA
S S
CH2
CH2
CH2
CH2
COOH
CH3
O
H
CH3
O
Thiamine Pyrophosphate
S S
CH2
CH2
CH2
CH2
COOH
Thiamine Pyrophosphate
+
+
FAD
E3 - Dihydrolipoamide Dehydrogenase
S S
CH2
CH2
CH2
CH2
COOH
H H
FAD
S
CH2
CH2
CH2
CH2
COOH
S
FADH2
+
+
PDH - Overall
Organic arsenicals are potent inhibitors of lipoamide-containing enzymes such as Pyruvate Dehydrogenase.
These highly toxic compounds react with “vicinal” dithiols such as the functional group of lipoamide.
HS
HS
R
S
S
R
R' As O AsR'+
H2O
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.
PDH - Regulation
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
22 FADH 2 NADH 6 ATP 2 CO 4 2AcCoA
Per glucose that entered glycolysis:
Thus, at the end of the cycle, we will have converted our glucose completely to CO2.
O H6 CO 6 O 6 OHC 2226126
We still won’t have used any oxygen or made any water.
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
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
Isocitrate Dehydrogenase Go’ = -20.9 kJ/mol
•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 enzyme3.Succinate leaves as a product4.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.
1.) Citrate Synthase 6.) Succinate Dehydrogenase2.) Aconitase 7.) Fumarase3.) Isocitrate Dehydrogenase 8.) Malate Dehydrogenase4.) α-Ketoglutarate Dehydrogenase 9.) Overall reaction5.) Succinyl-CoA Synthetase
Go’
G
Reaction EnzymeG°'(kJ/mol)
1 Citrate synthase -32.22 Aconitase +6.33 Isocitrate dehydrogenase -20.9
4 a-Ketoglutarate dehydrogenase complex -33.5
5 Succinyl-CoA synthetase -2.96 Succinate dehydrogenase 0.07 Fumerase -3.88 Malate dehydrogenase +29.7
Krebs Cycle Energetics
The citric acid is regulated by three simple mechanisms.1. Substrate availability2. Product inhibition3. Competitive feedback inhibition.
The Krebs cycle is amphibolic – intermediates are also used to make stuff.