Trypsin

Trypsin is a digestive protease produced in the pancreas (it is a component of pancreatic juice, which is released in the duodenum during digestion). Its function is to cleave dietary proteins, and for this it recognizes amino acids with basic side chains (lysine and arginine) and cleave peptide bonds in which they are involved. In order to be inactive in the cells that produce it (if this were not the case it would begin to degrade our own proteins), it is synthesized as trypsinogen. Trypsinogen is the zymogen of trypsin, an inactive form of an enzyme, characterized by having more amino acids than those that are required for the enzyme to be in its functional form. The idea is that these additional amino acids block the catalytic activity (e.g., preventing access of substrate to the active site). 

Once in the intestine, trypsinogen is proteolytically cleaved by the action of an enzyme called intestinal enteropeptidase. From the moment that a molecule becomes active trypsin, itself can begin to activate (by proteolytical cleavage) all other zymogens, not only trypsin but also chymotrypsin, carboxypeptidases and aminopeptidases. Thus, trypsin has a central role in the activation of digestive proteases, so it is necessary to ensure that any molecule, under normal conditions, will not become catalytically active inside the cells. The first line of protection is the synthesis of the enzyme in the form of trypsinogen. Additionally, cells which produce pancreatic trypsin also have a second line of defense, which involves the production of an inhibitory protein called pancreatic trypsin inhibitor. Therefore, even if spontaneously one trypsinogen acquires activity inside the cells, the presence of this inhibitor will prevent it to exerts its function and consequently, avoiding the cell to begin to cleave and activate other zymogens proteins.

Animation about trypsinogen activation

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Animation about the activation of trypsin

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Animation about enzyme catalysis

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.

Video about enzymes

Video about enzymes

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.

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 enzyme catalysis

This music is about enzyme catalysis and is an adaptation made by Dr. Ahern (www.davincipress.com/metabmelodies.html) of the song Close to You.

http://www.mediafire.com/?omzv9utktseeodu

Catalyze
My enzymes
Truly are inclined
To convert
Things they bind
Turn the key
Covalently
Cat-a-lyze
 

How do cells

Regulate these roles?
Allo-ster-ic controls
Two forms, see
States R and T
Mod-u-late
 
Competing inhibition keeps
The substrates from the active site
They raise Km, but leave Vmax and shirk
While the non-competers bind elsewhere
And lift the plot made on Lineweaver-Burk

Other ways
Enzymes can be blocked
When things bind
Then get locked
Stuck not free
Tied to the key
Su-i-cide

Penicillin’s action stops
Peptidoglycan cross-links in
Bacterial cell walls in awesome ways
Beta lactam ring’s reactive site
Starts bonding with D-D-transpeptidase
 
So there are
Several enzyme states
Counteract-ing substrates
Now you see
Blocking the key
Regulates
 
Cat-a-lysts
Have to be controlled
Some get slowed
Put on hold
It's sublime
How the enzymes
(slow) Cat-a-lyze
 
ahhhhhhhhhhhhhhhhhhh - cat-a-lyzeahhhhhhhhhhhhhhhhhhh - cata-
lyzeahhhhhhhhhhhhhhhhhhh - cat-a-lyze

Music about enzymes

Here it goes a link to download another Metabolic Melody from Dr. Ahern (www.davincipress.com/metabmelodies.html), this time about enzymes. It was base on the song Downtown.

http://www.mediafire.com/?w0gd96oo6ecpymg

Enzymes

Reactions alone

Energy peaks

Are what an enzyme defeats

In its catalysis

Enzymes

Transition state

Is what an enzyme does great

And you should all know this

Enzymes

Catalytic action won't run wild - don't get hysteric

Cells can throttle pathways with an enzyme allosteric

You know it's true

So when an effector fits

It will just rearrange

all the sub-u-nits

Inside an

ENZYME!

Flipping from R to T

ENZYME!

Slow catalytically

ENZYME!

No change in Delta G

(Enzyme, enzyme)

You should relax

When seeking out the Vmax though

There are many steps

Enzymes

Lineweaver Burk

Can save a scientist work

With just two intercepts

Enzymes

Plotting all the data from kinetic exploration

Let's you match a line into a best fitting equation

Here's what you do

Both axes are inverted then

You can determine Vmax and

Establish Km for your ENZYMES!

Sterically holding tight

ENZYMES!

Substrates positioned right

ENZYMES!

Inside the active site

Enzymes (Enzymes, enzymes, enzymes)

Could starve your cells to the bone

Thank God we all produce

Enzymes

Units arrange

To make the chemicals change

Because you always use

Enzymes

Sometimes mechanisms run like they are at the races

Witness the Kcat of the carbonic anhydrases

How do they work?

Inside of the active site

It just grabs onto a substrate

and squeezes it tight

In an

ENZYME!

CAT-al-y-sis

In an ENZYME!

V versus S

In an

ENZYME!

All of this working for you

(Enzyme, enzyme)

Glycolysis (enzymes of the payoff phase)

The payoff phase, as I mentioned earlier, concerns the whole of the last five reactions of glycolysis and allows the cell to obtain energy in this process. Here are some ideas on the enzymes of phase 5 payoff ...
6^th enzyme – Glyceraldehyde-3-phosphate dehydrogenase

This enzyme, often abbreviated to GAPDH, is presented in the form of a tetramer. Each subunit has about 35.9 kDa (331 amino acids) and shows how a molecule of cofactor NAD+. The subunits are designated by O, P, Q and R and are independent of each other. That is, each subunit catalyses the reaction without the intervention of others. As described in the post about the reactions of the payoff phase, the reaction catalyzed by this enzyme is a double one, involving an oxidation and an addition of a phosphate group. It is an enzyme that may be affected by the presence of arsenic in the body, causing the yield of glycolysis to become null. Its mechanism of action involves both a covalent catalysis and acid-base. To do this, it is essential the participation of cysteine ​​149 and histidine 176 for both types of catalysis, resectivamente. The substrate binds covalently to cysteine​​, forming a hemitioacetal. The laboratory level this enzyme is widely used (I also use ...) as a positive control techniques such as immunoblotting or RT-PCR, because in general their expression is constant in almost all cell types. So it is possible to determine changes in the expression of a certain gene or in the presence of a given protein by comparing it with the levels of GAPDH.

7^th enzyme – Phosphoglycerate kinase

This enzyme requires Mg²⁺ to make its catalytic activity. The name derives from the reaction of the enzyme in the reverse direction, which occurs during photosynthetic CO₂ fixation. It is responsible for the production of the first molecules of ATP in glycolysis. Its amino acid sequence has to be extremely conserved in different organisms. The monomeric enzyme is composed of two domains of equivalent size, which corresponds to half N-and C-terminal. The substrate (1,3-bisphosphoglycerate) binds to the first half, while ADP binds to the second. Presents a sequential kinetic mechanism in which catalysis occurs by a proximity effect.
8^th enzyme 8th – Phosphoglycerate mutase

The phosphoglycerate mutase is dimeric, with each of its subunits with about 32kDa. As the name implies, this is a mutase enzyme, ie, catalyzes the transfer of phosphoryl groups within a molecule. In other words, it changes the position of phosphoryl groups. In fact, the enzyme is phosphorylated (fosfoenzima is one), and will give up its phosphoryl group to the carbon of the substrate 2, resulting in an intermediate with two phosphoryl groups (2,3-bisphosphoglycerate). Only after this step, is that the phosphoryl group that was originally in the substrate (position 3) is removed, regenerating the initial form (phosphorylated) enzyme.

The phosphoglycerate mutase has three different isoforms (isozymes or isoenzymes), predominantly found in cardiac muscle, skeletal muscle and the third one in the other tissues.

9th enzyme – Enolase

The enolase is a dimeric metalloenzyme, and each subunit has about 40-50 kDa. These subunits have an antiparallel orientation, interacting with each other via two salt bridges, involving an arginine and a glutamate each. The N-terminal domain of alpha-3 subunit has four helices and beta sheets. The C-terminal domain has two beta sheets and two alpha-helices, and it ends with a barrel consists of beta sheets and alpha helices alternate. The two Mg²⁺ ions required for catalytic activity are critical in neutralizing negative charges. This enzyme has a pH optimum of about 6.5, and can also be called fosfopiruvato dehydratase. It was initially discovered in 1934 by researchers Lohmann and Meyerhof. As with the enzyme before the enolase also has three different isoforms, of which one is predominantly found in muscle tissue, the other in neurons and the third one in the remaining parts of the body.
The enolase is inhibited by fluoride ion, and this fact is exploited, for example, when collecting blood samples for analysis. In this case, when it is important to inhibit glycolysis (to keep unchanged the concentration of serum glucose), blood can be collected in tubes containing fluoride.
10^th enzyme – Pyruvate kinase

This enzyme is responsible for the second ATP production in glycolysis and is the third regulatory enzyme of this pathway. It needs the presence of two metal ions: K⁺ and Mg²⁺ (or Mn²⁺). It has four different isoforms, one located predominantly in the liver, another in red blood cells, the other in cardiac and skeletal muscle and brain and the latter is mainly found in fetal tissues. It is a tetrameric enzyme, each subunit has about 500 amino acids.

Main bibliographic sources:

- Voet D, Voet JG, Biochemistry, Wiley

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers