Link Reaction

Link reaction and other multi-enzyme complex catalyzed reactions

This post covers:
a) What are multi-enzyme complexes?
b) Link reaction
c) Other alpha ketoacid decarboxylations

Beta ketoacids undergo decarboxylation readily, however, alpha ketoacids do not. Refer to this blog post for a more detailed explanation on decarboxylation reactions.

Multienzyme complexes are the evolutionary future.

A multi-enzyme complex is a group of enzymes that are closely related to one another in structure and in function. They are organized such that the product of one enzymatic reaction can be directly channelized to the active site of the next enzyme for which it is the substrate. More formally a multienzyme complex can be defined as follows:

A multienzyme complex contains several copies of one or several enzymes or polypeptide chains packed into one assembly. Multienzyme complex carries out a single or a series of biochemical reactions taking place in the cells. It allows to segregate certain biochemical pathways into one place in the cell. -Wikipedia

Biochemically, the rate of a reaction is limited by the time it takes the enzyme to find its substrate in the solution. As successive enzymes in a multienzyme complex are close to one another physically, and the metabolites are channeled from one active site to the next, this time is greatly reduced. The channeling of metabolites from one enzyme to the next also successfully minimizes the enzyme’s side reactions with other metabolites. In this way, compartmentalization is achieved without the necessity for permeability barriers.

All the enzymes of a multienzyme complex are synthesized by a single gene. Thus, they can be coordinately synthesized and controlled.

Dissecting the link reaction.

Pyruvate, the end product of glycolysis is transported into the mitochondrion by a proton symporter. The mitochondria is the site of both the link reaction and the citric acid cycle. The link reaction is the irreversible route from glycolysis to the citric acid cycle.

In biochemistry, decarboxylation reactions are irreversible. Examples include the link reaction, 6-phosphogluconate dehydrogenase catalyzed reaction in the pentose phosphate pathway.

The link reaction converts pyruvate into acetyl coenzyme A (CoA), one of the substrates for the first reaction of the citric acid cycle. This reaction is catalyzed by the pyruvate dehydrogenase multienzyme complex (PDC).

$pyruvate + CoA + NAD^+\xrightarrow{\text{PDC}} acetyl-CoA + NADH + H ^+ +CO_2$

The above conversion occurs in five sequential steps catalyzed by the three enzymes of the pyruvate dehydrogenase complex: pyruvate dehydrogenase ($E_1$), dihydrolipoyl transacetylase ($E_2$) and dihydrolipoyl dehydrogenase ($E_3$).

The final products are highlighted in yellow.

• Pyruvate dehydrogenase requires thiamine pyrophosphate (TPP) as a cofactor. It catalyzes the conversion of pyruvate to hydroxyethyl$-$TPP (#1).
• The second enzyme, dihydrolipoyl transacetylase requires lipoamide as a co-factor. The hydroxyethyl$-$TPP carbanion attacks one of the sulphur atoms of the $S-S$ bond of lipoamide. This reaction results in the formation of acetyl-dihydrolipoamide (#2). Dihydrolipoyl transacetylase ($E_2$) then catalyzes the transfer of the acetyl group from acetyl$-$dihydrolipoamide to coenzyme A to form acetyl$-$CoA and dihydrolipoamide (#3).
• The last enzyme, dihydrolipoyl dehydrogenase ($E_3$) uses two cofactors: FAD to which it is covalently bound and NAD$^+$. Dihydrolipoyl dehydrogenase oxidizes dihydrolipoamide back to lipoamide. This results in a simultaneous reduction of the enzyme. The covalently bound FAD oxidizes the enzyme back to its original state via the formation of FADH$_2$. Finally, NAD$^+$ gets reduced to NADH + H$^+$ while oxidizing the FADH$_2$ back to FAD. Thus, by the end of the reaction both the enzyme $E_3$ and FAD are back in their original forms and can be reused. The only reduced product, NADH + H$^+$ enters the electron transport chain to get re-oxidized to NAD$^+$ for reuse.

Refer to:
this blogpost for details on TPP mechanism
this blogpost for lipoamide mechanism

PDC is regulated by the regulation of its first enzyme, pyruvate dehydrogenase (PDH).

Pyruvate dehydrogenase is regulated by feedback inhibition from its products: NADH and acetyl$-$CoA (figure A). It is also regulated by phosphorylation. In the phosphorylated form (PDH-b), this enzyme is inactive and in the dephosphorylated form (PDH-a), the enzyme is active.

‘[X]’ indicates concentration of substance X. Red arrows indicate inhibition and blue arrows indicate promotion of enzyme and thereby the reaction.

Understanding figure B

When the ratio of product to substrate of the link reaction is high, it means that the reaction is in progress. This means that the enzyme pyruvate dehydrogenase is in its active form (PDH-a). Thus, an accumulation of these products promote the conversion of the active PDH-a into the inactive PDH-b. On the other hand, insulin causes cellular uptake of glucose. This means that glucose, the substrate of glycolysis is entering the cell. Both glycolysis and link reaction must commence. Thus, insulin activates PDH phosphatase such that pyruvate dehydrogenase is converted to the active (PDH-a) form from the inactive (PDH-b) form. Now, PDH-a can begin to catalyze the link reaction. Note that the $\frac{[ATP]}{[ADP]}$ ratio can increase during fatty acid oxidation. This condition also promotes inactivation of pyruvate dehydrogenase.

Other alpha ketoacid decarboxylations occur similarly.

In addition to the PDC, most cells contain two other closely related multienzyme complexes: the $\alpha$-ketoglutarate dehydrogenase complex [also called 2-oxoglutarate dehydrogenase (OGDH), which catalyzes reaction 4 of the citric acid cycle and the branched-chain $\alpha$-keto acid dehydrogenase complex (BCKDH), which participates in the degradation of the amino acids isoleucine, leucine, and valine. These multienzyme complexes all catalyze similar reactions: the NAD$^+$linked oxidative decarboxylation of an $\alpha$-keto acid with the transfer of the resulting acyl group to CoA. All these enzyme complexes have the same $E_3$ enzyme and their $E_1$and $E_2$ subunits, though specific to their corresponding substrates, are homologous and use identical cofactors.

Arsenic poisoning was once an epidemic.

Source: Wikimedia commons

The Victorian era is known for its killer clothing. Arsenic dyes fabric bright green and so, it ended up in dresses, gloves, shoes, and artificial flower wreaths that women used to decorate their hair and clothes. Read more about mercury and arsenic fashion in this Nat Geo article.

Arsenic inhibits enzymes that catalyze $\alpha$-ketoacid decarboxylations by sequestering lipoamide. Thus, it is harmful to living organisms. In the Victorian era, before the dangers of arsenic more commonly realized, usage of arsenic to dye cotton, linen and paper was the order of the day. Hawksley’s new book, Bitten by Witch Fever, tells the story of the extensive use of arsenic in the 19$^{th}$ century. It includes pictures of objects and artworks made from substances that incorporated arsenic, and advertisements for arsenic-filled products for Victorian women, such as soap with a doctor’s certificate to ensure its harmlessness. Needless to say this resulted in a devastating amount of death and sickness.

Sources

• Arsenic poisoning in the Victorian era
• Read more about the symptoms of arsenic poisoning here.
• Harper’s Illustrated Biochemistry 31st Edition
• Biochemistry textbook by Donald Voet and Judith G. Voet