Cartoon about lab experiments

Krebs cycle (enzymes) - part 1

The Krebs cycle is a metabolic pathway composed of 8 biochemical reactions, each catalyzed by a different enzyme. Here is some information about the first four enzymes of the Krebs cycle ...

Citrate synthase

The citrate synthase is an enzyme widely used as a biomarker for the presence of intact mitochondria in cell cultures or organelle preparations. Despite being a mitochondrial enzyme it is encoded by nuclear DNA and synthesized in the cytosol.

This enzyme is the first regulatory enzyme in the Krebs cycle. It uses two different substrates, the acetyl-CoA and oxaloacetate. The oxaloacetate firstly binds to the enzyme, which induces conformational changes that create the binding site for the acetyl-CoA molecule. 

From a structural point of view, it is composed of 437 amino acid residues and has two subunits, each with about 20 alpha helices. The active center has three amino acid residues essential for the catalytic function of the enzyme, due to the establishment of specific interactions with the substrates - His274, His320, and Asp-375.

 

Its mechanism of action involves an aldol condensation. To view a video about the mechanism of action of citrate synthase, click here.

Aconitase

The aconitase is an enzyme that has a functional iron-sulfur cluster [Fe₄S₄]²⁺, which interacts with three cysteine ​​residues of the enzyme. It is especially sensitive to oxidative stress and, in particular, to superoxide anion, due to the iron-sulfur cluster. 

It has two homologues in our body, the iron-responsive element-binding protein (IRE-BP) and the 2-isopropylmalate dehydratase (or alpha-isopropylmalate isomerase).

From a structural standpoint, the aconitase has two conformations, one for the inactive and one for the active state. In the inactive form, it has four domains, the first three establish interactions with the iron-sulfur cluster, while the latter has the active center. When it becomes active, the enzyme is altered in the iron-sulfur cluster (Fe₃S₄ turns in Fe₄S₄), and this represents the main difference between the two conformations of the enzyme.

Its mechanism of action relies on a mechanism of dehydration-hydration, via the intermediate cis-aconitate.

Its active site has two amino acid residues particularly important for catalytic activity - His101 and Ser642.

The importance of this enzyme, in a physiological point of view, is supported by the existence of many diseases that affect it. One is referred to as aconitase deficiency. It is caused by a mutation in the gene that codes for a protein responsible for the assembling of the iron-sulfur cluster. This disease causes myopathy and exercise intolerance, because the aerobic catabolism of these individuals is compromised. Another disease is Friedreich's ataxia (FRDA), characterized by a lower activity of aconitase and other Krebs cycle enzyme, the succinate dehydrogenase. Besides these, there are studies that suggest a possible relationship between aconitase and diabetes. However, it is still an hypothesis that has to be best characterized.

 
Isocitrate dehydrogenase

Isocitrate dehydrogenase is the second regulatory enzyme in the Krebs cycle. There are three different isoforms of isocitrate dehydrogenase. One exists only in the mitochondrial matrix and uses NAD⁺ as the acceptor of electrons. The other isoforms use NADP⁺ as the acceptor of electrons and appear to have as main function the formation of NADPH, essential for the reducing anabolic reactions. These forms are present in the mitochondrial matrix, the cytosol and in the peroxisome. 

The forms using NADP⁺ as a cofactor have an homodimeric structure, while the one that uses NAD⁺ is a heterotetramer.

The reaction catalyzed by isocitrate dehydrogenase involves the formation of an intermediary, the oxalossuccinate.

From the clinical point of view, some mutations were found in isocitrate dehydrogenase in some brain tumors, including astrocytoma, oligodendroglioma and multiforme glioblastoma. There are also some studies that indicate a possible relationship between mutations in the enzyme and acute myeloid leukemia.

Alpha-ketoglutarate dehydrogenase

This is the third (and last!) regulatory point of the Krebs cycle.

This enzyme, which can also be referred to as oxoglutarate dehydrogenase, is actually a multienzyme complex. It consists of the following enzymes: alpha-ketoglutarate dehydrogenase, dihydrolipoyl succinyltransferase dihydrolipoyl dehydrogenase. It has a structure and a reaction mechanism very similar to the pyruvate dehydrogenase complex. Because of this, it is believed that possibly both complexes had a common origin and at some point of evolution they suffered a divergent evolution.

Clinically, this enzyme complex functions as an autoantigen in primary biliary cirrhosis, a form of acute hepatic failure. Moreover, its catalytic activity is also decreased in various neurodegenerative diseases.

Music about ATP

Who could imagine that the music Yellow Submarine, from The Beatles, could be transformed in a song about ATP? Dr. Ahern (www.davincipress.com/metabmelodies.html) does!

Click here to download the music

We All Need Just a Little ATP

Instructor sings
In the cells, inside of us, there's a sugar on adenine
Which is linked, to phosphate groups, and you know it as ATP

Everyone Sings:
If we build up a lot of ATP, we've too much energy, metabolically
If we build up a lot of ATP, we've too much energy, metabolically

Instructor sings
And the cellular decree, calls for storing up the energy
So we save, it chemically, building acids onto ACP

Everyone Sings:
Making fat stores a lot of energy, creates NADP, and uses ATP
Making fat stores a lot of energy, creates NADP, and uses ATP

Instructor sings
When we need, some energy, we burn fats in fancy cell machines
Acids all, get shuttled in, on the backs of little carnitines

Everyone Sings:
We break acids every hour today, in mitochnodri-ay, to acetyl-CoA
We break acids every hour today, in mitochnodri-ay, to acetyl-CoA

Instructor sings
One more thing, about this tune, should be remembered by, all of you
Burning fat, converts a few, FADs to FADH2s

Everyone Sings:
NADH is a product too, that you can surely use, when NAD's reduced
NADH is a product too, that you can surely use, when NAD's reduced

Animation about gas exchange in our body

Link to download the animation

Tattoo - steroid

Video about the action of insulin

Metabolic map on the synthesis of phospholipids in E. coli

Cartoon about alchemy

Thanks for the link, Ferraz!
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