Krebs cycle (enzymes) - Part 2

After a “troubled” end of school year (as always ...) and a vacation offline period, I am back to the posts. :)
In this post I will continue to describe the main characteristics of the Krebs cycle enzymes...

Succinyl-CoA synthetase
This enzyme, also called tiocinase succinate or succinate-CoA ligase, presents in its composition two subunits (alpha and beta). The alpha subunit binds to the CoA molecule, while the beta subunit binds GDP. There is an isoform of the enzyme (also mitochondrial) which has beta subunit with affinity to ADP, instead of GTP.

 

The mechanism of reaction occurs in three steps. The succinyl-CoA synthetase has a histidine residue which plays a central role in the transfer of the phosphate group to the biphosphate nucleotide that is bound to the beta subunit.

Failures in succinyl-CoA synthetase are the cause of the disease "fatal child lactic acidosis," which is a disease characterized by the production of high levels of lactic acid (which is easily understanded because of the Krebs cycle is a step of carbohydrates aerobic catabolism), which would normally cause death of the individual within the first 4 days of life.

Succinate dehydrogenase
This enzyme, also called succinate-coenzyme Q reductase, belongs simultaneously to the Krebs cycle and the mitochondrial respiratory chain, where is known as complex II. Because of this, it is the only Krebs cycle enzyme that is associated with the inner mitochondrial membrane (all the others are present in the matrix ...). It uses as a cofactor FAD.

Structurally, it presents four subunits, two hydrophobic and two hydrophilic ones. The first two are a flavoprotein (SdhA) and an iron-sulfur protein (SdhB). SdhA is the subunit that binds covalently FAD and succinate, while SdhB is characterized by the presence of three iron-sulfur clusters ([2Fe-2S], [4Fe-4S] and [4S-3Fe]). The hydrophobic subunits (SdhC and SdhD) function as membrane anchors. The two hydrophobic subunits form the cytochrome b, characterized by having six transmembrane domains, a heme group and a binding site for ubiquinone (which also involves subunit SDHB).

The binding site for succinate (subunit SdhA) involves the side chains of important amino acid residues, in particular Threonine254, Histidine354 and Arginine399.
The binding site for ubiquinone requires the presence of some essential amino acid residues, namely Proline160, Tryptophan 163, Tryptophan164, Histidina207 and Isoleucine209 (subunit B), Serine27, Isoleucine28 and Arginine31 (subunit C) and Tyrosine83 (subunit D).
Failures in the succinate dehydrogenase can lead to the appearance of several pathologies, including:
- Leigh syndrome, mitochondrial encephalopathy and optic atrophy (mutations in SdhA).
- Hereditary paraganglioma, hereditary pheochromocytoma and excessive production of superoxide ions (mutations in SdhB, SdhC and/or SdhD).

Fumarase
This enzyme, also known as fumarate hydratase or malate hydrolyase, has two isoforms, one mitochondrial and other cytosolic. It is a tetrameric enzyme, and the substrate binding site is called the catalytic center A and involves amino acid residues from three different subunits.

The enzyme is present in two forms, E1 and E2. The first is characterized by two acid/base groups (essential for its catalytic activity) without charge, being responsible for binding to the fumarate and subsequent chemical transformation in malate. The form E2 has the two acid/base groups in the ionized form of zwitterion (one with positive charge and one with negative charge), characterized by binding to malate. Both forms are interconverted during the catalytic cycle of the enzyme.

Deficiency in fumarase is called polyhydramnios and is also associated with the appearance of skin and uterus leiofibromyomas and renal carcinoma.

Malate dehydrogenase
The malate dehydrogenase has two distinct isoforms, a mitochondrial one (isoform 2) and other cytosolic (isoform 1). It is an enzyme which not only plays a role in Krebs cycle, but it is also involved in gluconeogenesis.

Structurally, it has similarities to lactate dehydrogenase, with a homodimeric structure (subunits with masses of 30-35 kDa). Each subunit has two domains, the first of which is characterized by a beta-sheet structure, while the other represents the binding site to NAD+, composed of four beta-sheet and one alpha helix. The subunits interact with one another through hydrogen bonding and hydrophobic interactions.
The active site of the enzyme is essentially hydrophobic, with separate binding sites for malate and NAD+. It presents some particular amino acid residues important for its catalytic activity, namely the Arginine102, Arginine109, Aspartate168, Arginine171 and Hystidine195.