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.