Citric acid cycle Ch. 16

Sample problem Entry into the citric acid cycle requires pyruvate conversion to acetyl CoA. The first step in this transformation is decarboxylation. What coenzyme do you suspect is involved? 5/4/2019 S.A. McFarland © 2007

TPP aids decarboxylation of a-keto acids 5/4/2019 S.A. McFarland © 2007

Pyruvate dehydrogenase (PDH) complex An example of a multienzyme complex Multienzyme complex: group of noncovalently associated enzymes that catalyze two or more sequential steps in a metabolic pathway Virtually all organisms contain multienzyme complexes, which represent a step forward in the evolution of catalytic efficiency Enhanced reaction rates due to smaller diffusion distances Side reactions minimized when intermediates are channeled between successive enzymes Reactions can be coordinately controlled 5/4/2019 S.A. McFarland © 2007

Pyruvate dehydrogenase (PDH) complex Electron micrograph of E. coli multienzyme complex ~4600 kD particle Diameter ~300 Å Complex contains multiple copies of three enzymes Pyruvate dehydrogenase (E1) Dihydrolipoyl transacetylase (E2) Dihydrolipoyl dehydrogenase (E3) Core of 24 E2 proteins arranged in a cube surrounded by 24 E1 proteins and 12 E3 proteins 5/4/2019 S.A. McFarland © 2007

Pyruvate dehydrogenase (PDH) complex Structural organization of E. coli multienzyme complex 60 subunit complex 24 E2 proteins (green spheres) arranged in a cube surrounded by 24 E1 proteins (orange spheres) and 12 E3 proteins (purple spheres) E2 proteins (green spheres) are trimers at the corners of the cube) E1 proteins (orange spheres) are dimers along each edge E3 proteins (purple spheres) dimers on each face 5/4/2019 S.A. McFarland © 2007

Pyruvate dehydrogenase (PDH) complex In mammals, yeast, and some bacteria, the PDH complex is larger and even more complicated! Numbers indicate 2-, 3-, 4-, and 5-fold axes of symmetry. The mitochodrially located ~10,000-kD eurkaryotic complex contains a dodecahedral core of 20 E2 trimers surrounded by 30 E1 a2b2 heterotetramers, and 12 E3 dimers, together with 12 or so copies of E3 binding protein, which facilitates E3 binding to the E2 core. Mammalian complexes also contain several copies of a kinase and a phosphatase that function to regulate the activity of the complex. Cubic E2 core Dodecahedral E2 core 5/4/2019 S.A. McFarland © 2007

Pyruvate dehydrogenase (PDH) complex 5/4/2019 S.A. McFarland © 2007

PDH complex Uses a total of five coenzymes 5/4/2019 S.A. McFarland © 2007

PDH complex 5 sequential reactions: pyruvate + CoA + NAD+ -> acetyl-CoA + CO2 + NADH 5/4/2019 S.A. McFarland © 2007

Lipoic acid Coenzyme that functions as an acyl group carrier Mixture of two structures: closed-ring disulfide (oxidized) form and an open-chain (reduced) form Oxidation-reduction cycles interconvert two forms Does not often occur free in nature, covalently attached to Lys via amide linkage Reduction potential of disulfide is -0.30 V (~20 kJ/mol higher in energy than dithiol) 5/4/2019 S.A. McFarland © 2007

Lipoic acid Coenzyme that functions as an acyl group carrier Lipoic acid (6,8-dithiooctanoic acid) was isolated and characterized in 1951. It took 10 tons of liver to yield 30 mg of lipoic acid. 5/4/2019 S.A. McFarland © 2007

PDH complex Conversion of pyruvate to acetyl-CoA (5 steps) 5/4/2019 S.A. McFarland © 2007

1. Pyruvate dehydrogenase (E1) Formation of hydroxyethyl-TPP (HETPP) Remember you’ve seen this reaction in alcoholic fermentation when pyruvate is decarboxylated to yield acetaldehyde. 5/4/2019 S.A. McFarland © 2007

Sample problem E1 of the PDH complex (pyruvate dehydrogenase) produces hydroxyethyl-TPP from pyruvate. What pathway that we’ve discussed involves the same type of reaction as the first step? What was the purpose of TPP? 5/4/2019 S.A. McFarland © 2007

2. Dihydrolipoyl transacetylase (E2) Formation of acetyl-dihydrolipoamide 5/4/2019 S.A. McFarland © 2007

2. Dihydrolipoyl transacetylase (E2) Formation of acetyl-dihydrolipoamide 5/4/2019 S.A. McFarland © 2007

3. Dihydrolipoyl transacetylase (E2) Transfer of acetyl group from acetyl-dihydrolipoamide to CoA 5/4/2019 S.A. McFarland © 2007

4. Dihydrolipoyl dehydrogenase (E3) Oxidation of dihydrolipoamide to complete catalytic cycle of E2 E3 contains a reactive Cys-Cys disulfide bridge and a tightly bound FAD Oxidation of dihydrolipoamide is an interchange reaction 5/4/2019 S.A. McFarland © 2007

5. Dihydrolipoyl dehydrogenase (E3) Oxidation of sulfhydryl groups to complete catalytic cycle of E3 FAD oxidizes sulfhydryl groups to reform the disulfide FAD funnels electrons to NAD+, i.e., it functions more as an electron conduit than as a source of sink of electrons Note that NAD+ is not a strong enough oxidant to oxidize FADH2 in this case (FAD in protein is about 0 V). 5/4/2019 S.A. McFarland © 2007

Swinging arm Reaction intermediates channeled between E2 (core) and the E1 and E3 proteins on the outside Lipoamide has a length of ~14 Å Lipoyllysyl arm acts as a long tether that swings the disulfide group from E1 (where it picks up a hydroxyethyl group) to the E2 active site (where the HE group is transferred to CoA), and from there to E3 (where the reduced disulfide is reoxidized) 5/4/2019 S.A. McFarland © 2007

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Swinging arm Flexible Pro- and Ala-rich segment contributes to the mobility of the lipoyllysyl arm 5/4/2019 S.A. McFarland © 2007

Arsenic poisoning As(III) compounds such as arsenite and organic arsenicals bind to sulfhydryl compounds to form bidentate adducts If the sulfhydryl compound is a lipoamide-containing enzyme, respiration comes to a halt! 5/4/2019 S.A. McFarland © 2007

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1. Citrate synthase Synthases are lyases working in reverse 5/4/2019 Remember a lyase is an enzyme that catalyzes the cleavage of substrates in simple elimination reactions that are NOT oxidation-reduction reactions and are NOT coupled to ATP hydrolysis. Synthases belong to the lyase category; you will see that synthetases belong to the ligase category. Learn the terms now! 5/4/2019 S.A. McFarland © 2007

Sample problem The standard free energy change for the citrate synthase reaction is -31.5 kJ/mol. Normally, you might expect such a large -ΔG to be coupled with the synthesis of ATP. However, no ATP (or GTP) is produced during this step. Give a possible explanation. 5/4/2019 S.A. McFarland © 2007

1. Citrate synthase Oxaloacetate binding causes 18o rotation of smaller domain, which closes the cleft Conformational change creates binding site for acetyl CoA Sequential binding very important in preventing wasteful hydrolysis of acetyl CoA Formation of citryl CoA thioester is followed by hydrolysis In gram+ bacteria and eukaryotes, citrate synthase is a dimeric protein composed of 2 identical subunits. Each subunit of the mammalian enzyme has 2 distinct domains; a small, flexible domain on the outer surface and a larger domain that forms the core of the protein. 5/4/2019 S.A. McFarland © 2007

A similar conformational change occurs for which enzyme in glycolysis? 5/4/2019 S.A. McFarland © 2007

1. Enol of acetyl-CoA is generated in rate-limiting step 2. Citryl-CoA is formed in a concerted acid-base catalyzed step (one of few enzymes that can form a C-C bond without a metal ion cofactor 3. Citryl-CoA is hydrolyzed to citrate and CoA 5/4/2019 S.A. McFarland © 2007

Acetyl CoA thioester is near the active site for the hydrolysis of citryl CoA thioester Concentration of oxaloacetate may be very low relative to concentration of acetyl CoA Considering the reaction mechanism above, provide a rationale behind the sequential binding of acetyl-CoA after oxaloacetate. 5/4/2019 S.A. McFarland © 2007

2. Aconitase Reversible isomerization of citrate and isocitrate Stereospecific addition of H2O to cis-aconitate to form 2R,3S-isocitrate 5/4/2019 S.A. McFarland © 2007

2. Aconitase [4Fe-4S] iron-sulfur cluster holds substrate in a preferred conformation Iron-sulfur clusters usually participate in redox processes; aconitase is an intriguing exception. 5/4/2019 S.A. McFarland © 2007

2. Aconitase [4Fe-4S] iron-sulfur cluster Addition of H2O across the double bond of cis-aconitate could potentially form four diastereomers; but only one is formed. 5/4/2019 S.A. McFarland © 2007