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Department of Chemistry and Biochemistry 3300 University of Lethbridge

III. The Cycle

Biochemistry 3300 Slide 1 (combustion)

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

•Efficient aerobic process that requires oxygen and produces dioxide.

Energy from these reactions is used to synthesize ATP .

Involves the complete oxidation of glucose to carbon dioxide and .

Oxidation Number C atoms* (glucose) 4

C atom (CO2) 0

Biochemistry 3300 Slide 2 of , , and

Cellular respiration involves three main phases:

Phase 1

Carbon skeletons of organic fuel molecules are degraded to acetyl groups that are attached to acetyl-CoA

Biochemistry 3300 Slide 3 Catabolism of Proteins, Fats, and Carbohydrates

Cellular respiration involves three main phases:

Phase 1 Carbon skeletons of organic fuel molecules are degraded to acetyl groups that are attached to acetyl-CoA

Phase 2

Oxidation of acetyl groups in the

Biochemistry 3300 Slide 4 Catabolism of Proteins, Fats, and Carbohydrates

Cellular respiration involves three main phases: Phase 1 Carbon skeletons of organic fuel molecules are degraded to acetyl groups that are attached to acetyl-CoA

Phase 2 Oxidation of acetyl groups in the citric acid cycle

Phase 3 Electrons carried by NADH and FADH are funneled 2 into the respiratory chain.

Biochemistry 3300 Slide 5 Catabolism of Proteins, Fats, and Carbohydrates

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

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

- Majority of , and oxidation.

- Majority of the generation of these compounds and others.

Citric Acid Cycle is as it acts both catabolically and anabolically

Biochemistry 3300 Slide 6 History

By 1930, it was established that some compounds:

Carboxylic acids (, lactate) Dicarboxylic acids (succinate, malate, -ketoglutarate) and Tricarboxylic acids (citrate, isocitrate)

would stimulate O2 consumption and CO2 production when added to “minced” muscle

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

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

Biochemistry 3300 Slide 7 History

Subsequently, Martius & Knoop showed: Pyruvate and Oxaloacetate can form citrate non-enzymatically (requires peroxide and basic conditions).

Odd: oxaloacetate is the in their pathway!

And then the showed: Succinate is formed from fumarate, malate or oxaloacetate.

Odd: These appear to be the reverse reactions!

Citric Acid is a CYCLE !!

Biochemistry 3300 Slide 8 Catabolism of Proteins, Fats, and Carbohydrates

Glycolysis – TCA cycle – mitochondria (eucaryotes)

Biochemistry 3300 Slide 9 Citric Acid Cycle are in the

Substrates must cross both the outer and inner mitochondrial membrane

Biochemistry 3300 Slide 10 Mitochondrial Membrane

Particles visualized by EM are “large” complexes

Inner membrane is rich in large protein complexes

Biochemistry 3300 Slide 11

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.

Biochemistry 3300 Slide 12 Pyruvate is oxidized to acetyl-CoA and CO 2

Combined dehydrogenation and of pyruvate requires the sequential action of three different enzymes (E1, E2, E3) and five different coenzymes.

Biochemistry 3300 Slide 13 Pyruvate Complex (PDH)

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

E. coli yeast -- E 24 60 1 Dihydrolipoyl transacetylase -- E 24 60 2 Dihydrolipoyl dehydrogenase -- E 12 12 3

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

Lipoate is connected to E 2

Biochemistry 3300 Slide 14 Pyruvate Dehydrogenase Complex Requires Five Coenzymes

1) 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 and an .

Biochemistry 3300 Slide 15 Structure

Cryo-EM reconstruction of PDH from bovine

Biochemistry 3300 Slide 16 Structure

E consists of three types of domains linked by short polypeptide linkers. 2

Biochemistry 3300 Slide 17 Structure and Mechanism

Oxidative decarboxylation of pyruvate to acetyl-CoA. Step 1 is rate limiting and responsible for specificity. Biochemistry 3300 Slide 18 Structure and Mechanism

Decarboxylation of pyruvate and formation of acetyl lipoyllysine

Biochemistry 3300 Slide 19 Structure and Mechanism

Formation of Acetyl-CoA

Biochemistry 3300 Slide 20 Why such a complex set of enzymes?

1. Enzymatic reaction rates are limited by diffusion, with shorter distance between subunits in an , 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.

Biochemistry 3300 Slide 21 Compounds are Poisonous

OH HS S O- As + O- As OH HS S R R

As(III) compounds, such as arsenite (AsO 3-) and organic arsenicals, 3 are toxic because they covalently attach to sulfhydryl compounds.

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

Biochemistry 3300 Slide 22 Structure and Mechanism

Arsenite inhibits E3

Biochemistry 3300 Slide 23 Mechanism of Dihydrolipoyl Dehydrogenase. More complicated than expected:

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

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

3. Spectrum of the arsenite-inactivated enzyme (2.) indicates that its FAD 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 FADH2 is pale yellow.

Explanation ?

Biochemistry 3300 Slide 24 Mechanism of Dihydrolipoyl Dehydrogenase.

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

Biochemistry 3300 Slide 25 Mechanism of Dihydrolipoyl Dehydrogenase.

Arsenite target

Biochemistry 3300 Slide 26 Catalytic Cycle of Dihydrolipoyl dehydrogenase

Biochemistry 3300 Slide 27 Eight Steps of the Citric Acid Cycle

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Biochemistry 3300 Slide 28