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Metabolism – Intro to Metabolism. CH339K. Going back to the early lectures. Why the big D G o ’ for Hydrolyzing Phosphoanhydrides ?. Electrostatic repulsion betwixt negative charges Resonance stabilization of products pH effects. pH Effects – D G o vs. D G o ’. (D G in kcal/mol).

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why the big d g o for hydrolyzing phosphoanhydrides
Why the big DGo’ for Hydrolyzing Phosphoanhydrides?
  • Electrostatic repulsion betwixt negative charges
  • Resonance stabilization of products
  • pH effects
ph effects d g o vs d g o
pH Effects – DGo vs. DGo’

(DG in kcal/mol)


cellular d gs are not d g o s
Cellular DGs are not DGo’ s

DGo’ 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

activation with atp luciferin
Activation with ATP - luciferin

Excited state of oxyluciferin forms and decays

for those who prefer more detail
For those who prefer more detail

Excerpted from Baldwin, T. (1996) Structure4: 223 – 228,

just because it s cool
Just because it’s cool…

Tobacco seedling w/ cloned luciferase

Southeast Asian firefly tree

just because it s cool1
Just because it’s cool…

Firefly squid (Watasenia scintillans) of Toyama Bay, Japan

New Zealand glowworm (Arachnocampa) cave


Electrochemistry in review

1.10 V

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

redox table
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?

d e o and k eq
DEo and Keq

For an actual half reaction aA + ne-⇌aA-

For an actual redox reaction:

A+n + ne- ⇌ A

B ⇌ B+n + ne-

A+n + B ⇌ A + B+n


(Analagous to the relation between DG and DGo’)

d e o and k eq cont
DEo and Keq (cont.)

Atequilibrium, the two are equal:




Or (rearranging)

Dr. Ready gets to the Point!

d e o and d g o
DEo and DGo


But we already know:


Another Point!

nad reduction nicotinamide adenine dinucleotide
NAD+ Reduction(Nicotinamide Adenine Dinucleotide)

NAD+ is a common redox cofactor in biochemistry

coenzyme q
Coenzyme Q

Coenzyme Q is another electron carrier in the cell


An Example:

What is DGo’ for the

Oxidation of NADH by Ubiquinone?

organic healthy
“Organic” ≠ “Healthy”

Vomiting and nausea, diarrhea, Headaches, Difficulty breathing, Pallor, Sweating, Palpitations, Lisps, Stomach pains/cramps, Seizures, Weakness, Drooling, and - of course - Death



  • Energy (ATP)
  • Parts (amino acids, etc.)
  • Reducing Power (NADH, NADPH)





catabolism of glucose
Catabolism of Glucose

C6H12O6 + 6O2 → 6CO2 + 6H2O

DGo’ = -2870 kJ/mol

It takes 31 kJ/mol to make an ATP. Enough energy is available for making ~90 (theoretically)

an aside on diets
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
An aside on diets (cont.)


Fat: 1 gram = 9 calories

Protein: 1 gram = 4 calories

Carbohydrates: 1 gram = 4 calories

The diet values come from the DGo’ for oxidizing the various biomolecules.

interconversion of c 6 sugars
Interconversion of C6 Sugars



-7.3 kJ/mol



Amino Sugars

-0.4 kJ/mol


Fatty Acids



glucose catabolism part 1 glycolysis
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 c 6 sugars again
Interconversion of C6 Sugars (Again)



-7.3 kJ/mol




Amino Sugars

-0.4 kJ/mol

Phosphohexose isomerase


Fatty Acids



don t eat the toothpaste
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
aldolase reaction
Aldolase Reaction
  • The standard free energy , DGo,for the aldolase reaction is very unfavorable (~ +25 kJ/mol)
  • Under cellular conditions, the real free energy, DG, is favorable (~ -6 kJ/mol)
  • [G-3P] is maintained well below the equilibrium level by being processed through the glycolytic pathway

H8 in human erythrocyte PGM

overall reaction
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
When two things look alike…

…there can be a problem.

arsenate poisoning in part
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
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
  • 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

Remember these guys?

  • 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

Model of L. mexicana glyceraldehyde-3-phosphate dehydrogenase complexed with N6-(1-naphthylmethyl)-2¢-deoxy-2¢- (3-methoxybenzamido)-adenosine.


Binding mode of 2-amino-N6-(p-hydroxyphenethyl)adenosine to T. brucei phosphoglycerate kinase.

energetics of glycolysis
Energetics of Glycolysis

DGo values are scattered: + and -

DG in cells is revealing:

  • Most values near zero
  • 3 of 10 Rxns have large, negative DG (i.e. irreversible)
  • Large negative DG Rxns are sites of regulation!
hexokinase 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
  • 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
Fates of Pyruvate





(Yeast, no O2)

(Critters, no O2)


In the absence of O2, no further oxidation occurs. NADH builds up, and NAD+ has to be regenerated to continue glycolysis

yeasties alcohol dehydrogenase
Yeasties: Alcohol Dehydrogenase





glucose catabolism part 2 pyruvate dehydrogenase
Glucose Catabolism Part 2Pyruvate 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
e1 pyruvate dehydrogenase proper
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
e2 dihydrolipoamide transacetylase lipoic acid
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
coenzyme a
Coenzyme A
  • Thioesters are activated compounds
  • Coenzyme A is a common activator
  • Warhead of CoA is the thiol
    • Hence, abbreviated HS-CoA
dihydrolipoamide transacetylase
Dihydrolipoamide Transacetylase
  • Lipoamide is reduced
  • Accepts acyl unit from PDC / Thiamine PP
  • Transfers to CoA
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.

pdh regulation
PDH Regulation

Product inhibition byNADH&acetyl CoA:

  • NADH competes with NAD+ for binding to E3.
  • Acetyl CoA competes with CoA for binding to E2.
pdh regulation1
PDH - Regulation

Regulation by E1phosphorylation/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 Kinaseincreases 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.
overall reaction1
Overall Reaction

Per glucose that entered glycolysis:

Thus, at the end of the cycle, we will have converted our glucose completely to CO2.

We still won’t have used any oxygen or made any water.

  • 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

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
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

Isocitrate Dehydrogenase

DGo’ = -20.9 kJ/mol

Oxidation: NAD+ oxidizes the hydroxyl carbon of isocitrate

Decarboxylation: A Mn+2 bound to the enzyme stabilizes the intermediate

Protonation: Reforms the carbonyl to generate product

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


CoA is displaced by an Orthophosphate

The phosphate group is transferred to a Histidine residue on the enzyme

Succinate leaves as a product

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


The citric acid is regulated by three simple mechanisms.

1. Substrate availability

2. Product inhibition

3. Competitive feedback inhibition.