Lecture 19-The Citric Acid Cycle and the Glyoxylate Cycle

Provide the catabolic purpose of the citric acid cycle and list the net products obtained via catabolism of 1 acetyl CoA through the citric acid cycle

The citric acid cycle is a set of eightreactions in which an acetyl group(from acetyl CoA) is condensed withoxaloacetate, two CO 2 are lost, andoxaloacetate is regenerated.

• Each round of the citric acid cycle generates (net):

• three NADH

• one FADH 2

• one GTP or ATP

The citric acid cycle is the gateway to the aerobic metabolism of any molecule that can be transformed into an acetyl group or a component of the citric acid cycle.

Give rationales why: 1) there is a physical link between the citric acid cycle and the electron transport chain, 2) why oxygen is required for the citric acid cycle to function.

1. The physical link between the citric acid cycle and the electron transport chain (ETC) is that the cycle's high-energy products, NADH and FADH2, are used by the ETC to produce ATP.

2. Oxygen is required for the citric acid cycle because the ETC, which uses oxygen as the final electron acceptor, is needed to regenerate NAD+ and FAD from NADH and FADH2. Without the regeneration of these cofactors, the cycle cannot continue.

For each metabolic enzyme in the citric acid cycle, name the products and reactants(including cofactors & coenzymes), name the enzyme that catalyzes the step, classify the enzymatic step into the 6 enzyme classes, and determine if the step is reversible or irreversible

Citrate Synthase

-Ligase

-catalyzes the irreversible condensation of acetyl CoA and oxaloacetate to form citrate

-Citrate synthase uses acid-base catalysis through the intermediate, citryl-CoA

-Citryl CoA intermediate is source of energy for ligation

-Citrate synthase exhibits induced fit

- products: citrate

- reactants: acetyl CoA and oxaloacetate

- irreversible

Aconitase

-Lyase

-Aconitase, an iron-sulfur protein (also referred to as a non-heme iron protein),catalyzes the reversible formation of isocitrate from citrate. It catalyzes a dehydration followed by a hydration, in another great example of a lyase reaction

-reacants & products: citrate -> cis-Aconitase->D-isocitrate

- reversible

Isocitrate Dehydrogenase

-Oxidoreductase

-Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate, forming α-ketoglutarate and capturing high-energy electrons as NADH. The first of two CO 2 molecules released in the citric acid cycle is produced here

-products: α-ketoglutarate

-reactants: isocitrate & oxalosuccinate

-irreversible

α-Ketoglutarate dehydrogenase complex

-oxidoreductase

-α-Ketoglutarate dehydrogenase complex catalyzes the irreversible synthesis of succinyl CoA from α-ketoglutarate, generating NADH. Succinyl coenzyme A is formed by the oxidative decarboxylation of alpha-ketoglutarate.

-products: succinyl CoA

-reactants: α-ketoglutarate

-irreversible

* 5 carbons -> 4 carbons, as CO 2 is released, but we also form an energy-rich thioester bond!

α-Ketoglutarate dehydrogenase complex's mechanism is the same as...

the PDH complex

The α-Ketoglutarate dehydrogenase complex and the reactions are structurally andmechanistically similar to the pyruvate dehydrogenase complex. Moreover, the E 3component is identical in both enzymes, and both substrates are alpha-ketoacids

* 3 enzymes, 5 coenzymes, same mechanism, same enzyme classes, also irreversible, and will even be regulated similarly!

Succinyl CoA synthetase

-ligase

-A compound with high phosphoryl-transfer potential is generated from succinyl coenzyme A. Succinyl CoA synthetase catalyzes the cleavage of a thioester linkage and concomitantly forms ATP. The formation of ATP by succinyl CoA synthetase is an example of a substrate-level phosphorylation because succinyl phosphate, a high phosphoryl-transfer potential compound that is formed as an intermediate, donates a phosphate to ADP.

-products: succinate

-reactants: succinyl CoA

-reversible

Mechanism of Succinyl-CoA Synthetase

1. a phosphate group displaces CoA in succinyl-CoA. The product, succinyl phosphate, is an acyl phosphate, which releases a large amount of free energy when hydrolyzed.

2. succinyl phospahte donates its phosphoryl group to a His residue on the enzyme, producing a phospho-His intermediate and releasing succinate.

3. The phospho-histidine residue then swings over to a boundGDP (or ADP)

4. The phosphoryl group is transferred to form GTP (orATP)

Succinate dehydrogenase

-oxidoreductase

-Succinate dehydrogenase catalyzes the dehydrogenation of succinate to fumarate. This oxidation-reduction reaction requires an FAD prosthetic group, which is reduced toFADH 2 during the reaction. Succinate dehydrogenase is also known as Complex II ofthe electron transport chain

- products: fumerate

-reactants: succinate

-reversible

Fumarase

-lyase

-Fumarase catalyzes the next step, which is the hydration of fumarate to form L-malate. The hydration is a stereospecific trans addition of H+ and OH−. The OH− group adds to only one side of the double bond of fumarate; hence, only the L isomer of malate is formed

-products: L-malate

-reactants: fumerate

-reversible

Malate Dehydrogenase

-oxidoreductase

-Malate dehydrogenase catalyzes the oxidation of L-malate to form oxaloacetate. NAD+ is again the hydrogen acceptor. The standard free energy for this reaction, unlike that for the other steps in the citric acid cycle, is significantly positive. The oxidation ofmalate is driven by the use of the products—oxaloacetate by citrate synthase and NADH by the electron-transport chain

-products: oxalocetate

-reactants: L-malate

- reversible

Oxaloacetate is regenerated by...

the oxidation of succinate (steps 6, 7, 8)

Succinate dehydrogenase, fumarase, and malate dehydrogenase catalyze successive reactions to regenerate oxaloacetate. FADH 2 and NADH are generated. Oxaloacetate can condense with another acetyl CoA to initiate another cycle

Calculate the ATP equivalents obtained from the complete oxidation of key carbohydrates(glucose, lactose, sucrose, maltose, fructose, galactose), glycerol, alanine, and lactate

1 NADH = 2.5 ATP

1 QH2 (FADH2 ) = 1.5 ATP

32 ATP from 1 glucose

Identify the means by which the citric acid cycle is regulated by providing examples of both positive and negative regulators for each regulated step

the key control points in the citric acid cycle are the reactions catalyzed by citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase complex(the metabolically irreversible reactions).

Negative regulation (inhibitors):

between acetyl CoA and citrate

- citrate, succinyl CoA, NADH,

between isocitrate and α-Ketoglutarate

- ATP and NADH

between α-Ketoglutarate and succinyl CoA

- ATP, succinyl CoA, NADH

positive regulation (activators):

between isocitrate and α-Ketoglutarate

-ADP

Link the regulation of the citric acid cycle to the anabolic activities of the citric acid cycle by providing specific examples of anabolic reactions involving citric acid cycle intermediates.

Many of the steps of the citric cycle are reversible except the three regulated reactions (citrate synthase, isocitrate dehydrogenase, & a-ketoglutarate dehydrogenase).

Therefore, with constant input of acetyl CoA, reactions can be catabolic, producing NADH and FADH 2, or anabolic, using citric acid cycle intermediates to make more complex molecules.

Therefore, the citric acid cycle is an amphibolic pathway

Explain how the citric acid cycle intermediates are replenished after anabolic use by providing specific enzymatic anaplerotic reactions

Because the citric acid cycle provides precursors for biosynthesis, reactions to replenish the cycle components are required if the energy status of the cells changes. These replenishing reactions are called anaplerotic reactions. A prominent anaplerotic reaction is catalyzed by pyruvate carboxylase, which synthesizes oxaloacetate by the carboxylation of pyruvate. Other anaplerotic reactions include transaminations from amino acids and malic enzyme

Describe the glyoxylate cycle found in plants and some bacteria and explain why animals cannot store carbons from fat as sugars, but plants and some bacteria can

The glyoxylate cycle is similar to the citric acid cycle but bypasses the two decarboxylation steps, allowing the synthesis of carbohydrates from fats. Succinate can be converted into oxaloacetate and then into glucose. The glyoxylate cycle, which occurs in organelles called glyoxysomes, is prominent in oil-rich seeds such as sunflower seeds

-Plants can make sugars from fats, but mammals cannot because the plants have a glyoxylate cycle in addition to citric acid cycle.

Isocitrate Lyase

Lyase

Reactant: Isocitrate

Products: Succinate and Glyoxylate

Cofactors / Coenzymes: None required

Irreversible

Bypasses the CO₂-releasing steps of the TCA cycle, conserving carbon skeletons for carbohydrate synthesis.

Malate synthase

ligase

Reactants:Glyoxylate, Acetyl-CoA, H₂O

Products:Malate, CoA-SH

Cofactors / Coenzymes: None required

irreversible

Incorporates another acetyl-CoA molecule into the cycle to form malate, which can be converted into oxaloacetate → glucose via gluconeogenesis.