1. Citric Acid Cycle

Pyruvate Dehydrogenase

This section is long and complicated. Fair warning.

Overview

Oxidative Phosphorylation (OP) provides 95% of the energy we consume. In OP most of the electrons driving the Electron Transport Chain are released during The Citric Acid Cycle bound to the universal electron acceptors, NADH+ and FAD. The Citric Acid Cycle itself is fed by Acetyl-CoA. This lecture covers the generation of Acetyl-CoA.

After glycolysis, a carrier protein transports pyruvate into the mitochondria. There, Acteyl-CoA is formed by oxidative decarboxylation (OD). That is, pyruvate is simultaneously “decarboxylated” and “oxidized.”

General Decarboxylation Reaction

Decarboxylation_reaction

If the reactant is pyruvate, then the R side-chain in the context of decarboxylation is the acetyl group on the left.

The conversion of pyruvate into Acetyl-CoA

 

pyruvate

Pyruvate Dehydrogenase Complex (PDH)

The conversion from pyruvate to Acetyl-CoA is catalyzed by the Pyruvate Dehydrogenase Complex (PDH), a multi-enzyme consisting of three subunits: E1, E2, and E3. The makeup of this complex various by species. Bacteria differs from Bovine differs from Human, for example. The PDH is large at 500 Angstrom, double the size of a ribosome.

The PDH requires five additional coenzymes

  • E1: Thiamine Phosphate (TPP)
  • E2: Coenzyme-A and Lipotate (lipoic acid)
  • E3: FAD and NAD+

Overview of Oxidative Decarboxylation

  1. Decarboxylation
  2. Oxidation
  3. Transfer
  4. Regeneration

This is a highly exergonic and irreversible process. Once converted, Acetyl-CoA is either  fully oxidized in the CAC or it gets converted into fatty acids and lipids.

A consequence of this irreversibility is that fats can not be converted into sugar. True in animals, at least: plants and some microorganisms have a unique pathway called the glyoxylate cycle that supports this conversion.

Oxidative Decarboxylation in Detail

Step 1: Decarboxylation

Catalyzed by E1, relies on (“implicates”) TPP.

TPP + Pyruvate —(E1)—> HTTP + CO2

The acidic carbon in TPP is deprotonated. CO2 is released from Pyruvate, thus removing one carbon. The remaining acetyl group from pyruvate bonds to TPP. TPP is reprotonated after released of CO2. This creates Hydroxyethyl-TPP (HTTP).

Step 2: Oxidation

Oxidation of HTTP. Catalyzed by E1, helped by lipoamide arm bound to E2.

HTTP + lipamide —(E1)—> TTP + Acetyl Lipoamide

Note that TPP is back to its old self again!

Acetyl Lipoamide has a thioester group. Thioesters have a high free energy of hydrolysis.  Unlike oxygen esters, the presence of sulfter in a thioester prevents resonance, which makes it less stable. Thus, the formation of thioester in Acetyl lipoamide serves to activate the acetyl group. That is, it sets up the transfer of the acetyl group from the lipoamide to Coenzyme-A.

Thiol: R-S-H (trigonal)

Step 3: Transfer

Catalyzed by E2. The lipoamide arm remains bound to E2

CoA-SH + Acetyl Lipoamide —(E2)—> Acetyl-CoA + dihydrolipoamide (fully reduced)

The disulfide bond in the lipoamide is reduced in Step 3. It has to be regenerated for the reaction to proceed. Otherwise the PDH complex would not continue functioning correctly.

Step 4: Regeneration

Catalyzed by E3. The lipoamide arm remains bound to E2

dihydrolipoamide —(E3)—> lipoamide + FADH2 –> FAD + NADH + H+

Note that FAD accepts two protons to become FADH2. Once the NADH is released into the mitochondria, it can be used for OD.

The lecture then goes into an involved slideshow showing the movement of these coenzyme and reaction sites across the PDH. Best just to review the slides there. The key take-aways should be summarized above though.

Regulation of PDH

Since the synthesis of Acetyl-CoA is the gateway to both OP and fatty acid synthesis, the PDH activity will depend on the energy state of the cell.

Specifically, PDH is allosterically inhibited by high energy states. But it’s more than just ATP. PDH is also inhibited by high concentration of reaction intermediates Acetyl-CoA and NADH, along with the reaction products, ATP and fatty acids. All four act as allosteric inhibitors of PDH. In particular, Acetyl-CoA acts on E2, while NADH acts on E3.

Similarly, it’s allosterically activated by low energy states. When ATP is needed, the substrate of the reaction, Coenzyme-A and NAD+ are at high concentrations, as is AMP (ATP less two phosphate groups). All are allosteric activators of PDH.

Second regulation: reversible phosphorylation

The two states of PDH, active and inactive, are also regulated by kinase/phosphatase enzymes. Recall that kinase adds a phosphate group, while phosphatase removes it. These  enzymes are Pyruvate Dehydrogenase Kinase (PDK) and Pyruvate Dehydrogenase Phosphatase (PDP), respectively.

PDK active => PDH inactive
PDP active => PDH active

When PDK is activated, a phosphate group binds to E1, rending PDH inactive. Similarly, PDP removes that phosphate to re-activate it again. The allosteric inhibitors of PDH, therefore, activate the kinase and put PDH in its inhibited state. That is, Acetyl-CoA, NADH, and ATP.

Similarly, the reaction substrates including Pyruvate and ADP, inhibit the kinase, preventing further inhibition.

In muscles, calcium released during muscle contraction active PDP, thus activate PDH.

Hormonal Control of PDH

In the liver and adipose tissue, epinephrine and insulin play a role in PDP activation.

Epinephrine in the liver causes an increase in the concentration of calcium in the liver. Calcium stimulates PDP, thus activates PDH.

Insulin also stimulates PDP. The idea being that too much glucose is present in the blood supply and thus needs to be catabolized.

 

 

 

 

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