III. Metabolism The Citric Acid Cyclepeople.uleth.ca/~steven.mosimann/bchm3300/Bchm3300_L6.pdf · Biochemistry 3300 Slide 1 III. Metabolism The Citric Acid Cycle Department of Chemistry

Biochemistry 3300 Slide 1

III. MetabolismThe Citric Acid Cycle

Department of Chemistry and BiochemistryUniversity of Lethbridge

Biochemistry 3300


Cellular Respiration (combustion)

Cellular respiration is the step-wise release of energy from glucose,fatty acids and (some) amino acids.

•Efficient aerobic process that requires oxygen and produces carbon dioxide.

Energy from these reactions is used to synthesize ATP molecules.

Involves the complete oxidationof glucose to carbon dioxide and water.

Oxidation NumberC atoms* (glucose) 4C atom (CO

2) 0


Catabolism of Proteins, Fats, and Carbohydrates

Phase 1

Carbon skeletons oforganic fuel moleculesare degraded to acetyl groups that are attachedto acetyl-CoA

Cellular respiration involvesthree main phases:



Phase 2

Oxidation of acetyl groupsin the citric acid cycle


Phase 1Carbon skeletons oforganic fuel moleculesare degraded to acetyl groups that are attachedto acetyl-CoA



Phase 3Electrons carried by NADHand FADH

2 are funneled

into the respiratory chain.

Phase 2Oxidation of acetyl groupsin the citric acid cycle

Phase 1Carbon skeletons oforganic fuel moleculesare degraded to acetyl groups that are attachedto acetyl-CoA




- Majority of carbohydrate, fatty acid and amino acid oxidation.

- Majority of the generation of these compounds and others.

Citric Acid Cycle is amphibolic as it acts both catabolically and anabolically

The citric acid cycle is alsocalled the Krebs cycle or the tricarboxylic acid (TCA) cycle.

The citric acid cycle is the“hub” of the metabolic system.


HistoryBy 1930, it was established that some compounds:

Carboxylic acids (acetate, lactate)Dicarboxylic acids (succinate, malate, -ketoglutarate) andTricarboxylic acids (citrate, isocitrate)

would stimulate O2 consumption and CO

2 production when added to

“minced” muscle

1935: Albert Szent-Gyorgyi Succinate → Fumarate → Malate → Oxaloacetate

Carl Martius & Franz KnoopCitrate [→ Cis-aconitate] → Isocitrate → -ketoglutarate →Succinate → Fumarate → Malate → Oxaloacetate


Subsequently, Martius & Knoop showed:

Pyruvate and Oxaloacetate can form citrate non-enzymatically (requires peroxide and basic conditions).

Odd: oxaloacetate is the product in their pathway!

And then Hans Krebs the showed:

Succinate is formed from fumarate, malate or oxaloacetate.

Odd: These appear to be the reverse reactions!

Citric Acid metabolic pathway is a CYCLE !!

History



Glycolysis – cytosolTCA cycle – mitochondria (eucaryotes)


Citric Acid Cycle enzymes are in the mitochondrial matrix

Substrates must cross both the outer and inner mitochondrial membrane


Mitochondrial Membrane

Particles visualized byEM are “large” protein complexes

Inner membrane isrich in large protein complexes


Nathan Kaplan and Fritz Lipmann discovered Coenzyme A (CoA)

Ochoa and Lynen showed that acetyl-CoA is an intermediate in the conversion of pyruvate to citrate.

Coenzyme A


Pyruvate is oxidized to acetyl-CoA and CO

2

Combined dehydrogenation and decarboxylation of pyruvate requires the sequential action of three different enzymes

(E1, E2, E3) and five different coenzymes.


Pyruvate Dehydrogenase Complex (PDH)

E. coli yeast

Pyruvate dehydrogenase -- E1 24 60

Dihydrolipoyl transacetylase -- E2 24 60

Dihydrolipoyl dehydrogenase -- E3 12 12

Molecular weight of 4,600,000 Da ; 50 nm in diameter

PDH complex contains three subunits, present in multiplecopies. Number varies among species.

Lipoate is connected to E2


Pyruvate Dehydrogenase Complex Requires Five Coenzymes

1) Nicotinamide adenine dinucleotide (NAD+)

2) Thiamin pyrophosphate (TPP)

3) Flavin adenine dinucleotide (FAD)

4) Coenzyme A (CoA)

5) Lipoate

The lipoyllysl moiety acts as a carrier of both hydrogen andan acetyl group.


Structure

Cryo-EM reconstruction of PDH from bovine kidney


Structure

E2 consists of three types of domains linked by short polypeptide linkers.


Structure and Mechanism

Oxidative decarboxylation of pyruvate to acetyl-CoA.

Step 1 is rate limiting and responsible for substrate specificity.



Decarboxylation of pyruvate and formationof acetyl lipoyllysine



Formation of Acetyl-CoA


Why such a complex set of enzymes?

1. Enzymatic reaction rates are limited by diffusion, with shorter distance between subunits in an enzyme, the substrate can be directed from one subunit (catalytic site) to another.

2. Channeling metabolic intermediates between successive enzymes minimizes side reactions. (Substrate channeling).

3. Local substrate concentration is kept high.

4. The reactions of a multienzyme complex can be coordinately controlled / regulated.


Arsenic Compounds are Poisonous

HS

HS

R

O- AsOH

OH

+

S

S

R

AsO-

As(III) compounds, such as arsenite (AsO3

3-) and organic arsenicals,

are toxic because they covalently attach to sulfhydryl compounds.

Vicinal (adjacent) sulfhydryls form bidentate adducts (top right)



Arsenite inhibits E3


Mechanism of Dihydrolipoyl Dehydrogenase.

More complicated than expected:

1. Spectra of oxidized dihydrolipoamide dehydrogenase (E3) isunaffected by arsenite.

2. NADH reaction with the oxidized enzyme in the presence ofarsenite → forms an enzymatically inactive species.

3. Spectrum of the arsenite-inactivated enzyme (2.) indicates that itsFAD prosthetic group is fully oxidized.

Recall: The oxidation state of the flavin in a flavoprotein is readily established from its characteristic UV-Vis Spectrum: FAD is intense yellow, whereas FADH

2 is pale yellow.

Explanation ?



Oxidized dehydrolipoamide dehydrogenase has an additional electron acceptor.

Arsenite inhibition suggests a disulfide as acceptor.

See X-Ray structure of dehydrolipoamide DH from P. putida, PDBID 1LVL

Catalytic active residues: Cys 43 & 48 , Tyr 181



Arsenitetarget


Catalytic Cycle of

Dihydrolipoyldehydrogenase


Eight Steps of the Citric Acid Cycle

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III. Metabolism The Citric Acid Cyclepeople.uleth.ca/~steven.mosimann/bchm3300/Bchm3300_L6.pdf · Biochemistry 3300 Slide 1 III. Metabolism The Citric Acid Cycle Department of Chemistry