Department of Chemistry and Biochemistry Biochemistry 3300 University of Lethbridge
III. Metabolism The Citric Acid Cycle
Biochemistry 3300 Slide 1 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 oxidation of glucose to carbon dioxide and water.
Oxidation Number C atoms* (glucose) 4
C atom (CO2) 0
Biochemistry 3300 Slide 2 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
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 citric acid cycle
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 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
Biochemistry 3300 Slide 6 History
By 1930, it was established that some compounds:
Carboxylic acids (acetate, 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 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 !!
Biochemistry 3300 Slide 8 Catabolism of Proteins, Fats, and Carbohydrates
Glycolysis – cytosol TCA cycle – mitochondria (eucaryotes)
Biochemistry 3300 Slide 9 Citric Acid Cycle enzymes are in the mitochondrial matrix
Substrates must cross both the outer and inner mitochondrial membrane
Biochemistry 3300 Slide 10 Mitochondrial Membrane
Particles visualized by EM are “large” protein complexes
Inner membrane is rich in large protein complexes
Biochemistry 3300 Slide 11 Coenzyme A
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 decarboxylation of pyruvate requires the sequential action of three different enzymes (E1, E2, E3) and five different coenzymes.
Biochemistry 3300 Slide 13 Pyruvate Dehydrogenase Complex (PDH)
PDH complex contains three subunits, present in multiple copies. Number varies among species.
E. coli yeast Pyruvate dehydrogenase -- 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) 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 and an acetyl group.
Biochemistry 3300 Slide 15 Structure
Cryo-EM reconstruction of PDH from bovine kidney
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 substrate 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 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.
Biochemistry 3300 Slide 21 Arsenic 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 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 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
NEXT
Biochemistry 3300 Slide 28