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Wednesday, January 09, 2008

Serine proteases Presence of Ser-His-Asp catalytic triad at the active site

030. A common feature of all serine proteases is:
1. Autocatalytic activation of zymogen precursor
2. Tight binding of pancreatic trypsin inhibitor
3. Cleavage of protein on the carboxyl site of serine residues
4. Presence of Ser-His-Asp catalytic triad at the active site
Answer
4. Presence of Ser-His-Asp catalytic triad at the active site
Reference:
Harper 26th Edition Page 53
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Discussion
  • The serine proteases are a family of enzymes that cut certain peptide bonds in other proteins.
  • This activity depends on a set of amino acid residues in the active site of the enzyme - one of which is always a serine (thus accounting for their name).
  • In mammals, serine proteases perform many important functions, especially in digestion, blood clotting, and the complement system.
  • Digestive Enzymes :
    • Three protein-digesting enzymes secreted by the pancreas are serine proteases:
      • chymotrypsin
      • trypsin
      • elastase
    • These three share closely-similar structures (tertiary as well as primary). In fact, their active serine residue is at the same position (Ser-195) in all three.
    • Despite their similarities, they have different substrate specificities; that is, they cleave different peptide bonds during protein digestion.
  • Clotting Factors
    • Several activated clotting factors are serine proteases, including
      • Factor 10 (X)
      • Factor 11 (XI)
      • Thrombin
      • Plasmin
  • Complement Factors
    • Several proteins involved in the complement cascade are serine proteases, including
      • C1r and C1s
      • the C3 convertases
        • C4b,2a
        • C3b,Bb
Explanation
1. As seen above the Serine proteases need not be limited to autocatalytic activation of zymogen precursor
2. Tight binding to pancreatic trypsin inhibitor is not a common feature
3. Serine protease means that the enzyme contains serine. It can cleave a variety of residues
4. Presence of Serine at position 195, histidine at position 57, and aspartate at position 102 make up what is often called the catalytic triad. The serine hydroxyl group is the essential catalytic group and is why this family is known as the serine proteases.
a. Step 1 : Once the substrate is bound, the hydroxyl group of serine 195 nucleophilically attacks the scissile peptide's carbonyl group. The hydroxyl group is made more nucleophilic by the neighboring histidine that extracts the hydroxyl hydrogen, a process that is facilitated by the polarizing effects of aspartate 102. A covalent bond forms between the serine and the substrate to yield a complex known as the tetrahedral intermediate. The tetrahedral intermediate, which resembles the reaction's transition state, is stabilized by two amide hydrogens that hydrogen bond to the anionic oxygen. This region is known as the "oxyanion hole" because it is occupied by the intermediate's oxyanion group.
b. Step 2 : The tetrahedral intermediate quickly decomposes back to a planar carbonyl group. This can happen in one of two ways. The bond to the serine can dissolve regenerating the original starting components (this is just the reverse of step 1), or the bond to the nitrogen can break, releasing the C-terminal portion of the substrate while forming an ester linkage to the N-terminal portion. This latter assembly is called the acyl-enzyme intermediate. Note that the hydrogen temporarily held by the histidine has now been passed on to the leaving polypeptide fragment.
c. Step 3 : Next, the ester bond of the acyl-enzyme must be broken. This is accomplished by another nucleophilic attack on the carbonyl group, this time by a water that has diffused into the active site. The water transfers one hydrogen to the histidine while forming a covalent bond to the carbonyl carbon. The result is another tetrahedral intermediate stabilized by the amide groups in the oxyanion hole.
d. Step 4 : In the last step, the tetrahedral intermediate decomposes by breaking the bond to the serine hydroxyl group. The hydrogen held by the histidine is transferred to the serine and the substrate is released with a carboxylic acid terminus. The enzyme is returned to its original state ready to bind the next protein.

Comments

Serpins :

Serpins are Serine Protease Inhibitors.

Here is a list of a few important serine proteases and the serpins that control them.

Serine Protease

Serpin

Chymotrypsin

alpha-1-antichymotrypsin

Complement factor C1s

C1 Inhibitor (C1INH)

Elastase (secreted by neutrophils)

alpha-1-antitrypsin

Clotting factor 10 (X)

antithrombin III

Thrombin

antithrombin III

Plasmin

alpha-2-antiplasmin

Trypsin

pancreatic trypsin inhibitor

How Serpins Work

The serpins inhibit the action of their respective serine protease by mimicking the three-dimensional structure of the normal substrate of the protease.

The serine protease binds the serpin instead of its normal substrate. This alone would block any further activity by the protease. But the serpin has another trick to play.

The protease makes a cut in the serpin leading to

the formation of a covalent bond linking the two molecules;

a massive allosteric change in the tertiary structure of the serpin;

which moves the attached protease to a site where it can be

destroyed.

Importance of Serpins

Almost 20% of the proteins found in blood plasma are serpins.

Their abundance reflects their importance: putting a stop to proteolytic activity when the need for it is over.

This is especially important for the

clotting and

complement

systems where a tiny initial activating event leads to a rapidly amplifying cascade of activity.

Serpin Deficiencies

A number of inherited human diseases are caused by a deficiency of a particular serpin. The deficiency usually results from a mutation in the gene encoding the serpin.

Examples:

Alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin inactivates the elastase secreted by neutrophils. When the lungs become inflamed, neutrophils secrete elastase as a defensive measure. However, it is important to inactivate this elastase as soon as its job is done. That is the function of alpha-1-antitrypsin.

(Its name, alpha-1-antitrypsin, suggests that it attacks the digestive enzyme, trypsin. In vitro, it does, but in the body, alpha-1-antitrypsin is found in the blood, not the intestine. Inactivation of trypsin in the intestine is the function of another serpin, pancreatic trypsin inhibitor.)

People with an inherited deficiency of alpha-1-antitrypsin are prone to emphysema. An effective treatment is on the horizon now that genetic engineering has produced sheep that secrete human alpha-1-antitrypsin in their milk. [More]

Alpha-1-antitrypsin deficiency can also lead to liver damage. Alpha-1-antitrypsin is synthesized in the liver. However, some mutant versions of the molecule form insoluble aggregates within the liver cells. This mechanism is similar to that of the prion diseases where protein aggregates destroy neurons in the brain. [Link]

C1INH deficiency

A deficiency of C1INH produces hereditary angioneurotic edema (HANE). Patients are at risk of occasional explosive triggering of the complement system. The massive release of anaphylatoxins (C3a, C5a) may cause dangerous swelling (edema) of the airways, as well as of the skin and intestine.

Antiplasmin deficiency

A deficiency in antiplasmin puts the person at risk of uncontrollable bleeding.

Antithrombin deficiency

A deficiency in antithrombin puts the person at risk of spontaneous blood clots, which can lead to a heart attack or stroke.

The Evolution of the Serine Proteases

The close sequence similarity of the various mammalian serine proteases suggests that each is the product of a gene descended by repeated gene duplication from a single ancestral gene.

Tips:

Other Serine Proteases

Serine proteases and molecules similar to them are found elsewhere in nature.

Subtilisin

Subtilisin is a serine protease secreted by the bacterium Bacillus subtilis. Although it has the same mechanism of action as the serine proteases of mammals, its primary structure and tertiary structure are entirely different.

Here, then, is an example at the molecular level of convergent evolution: two molecules acquiring the same function (analogous) but having evolved from different genes.

Acetylcholinesterase

This enzyme is built like and acts like the other serine proteases, but its substrate is the neurotransmitter acetylcholine, not a protein.

It is found at several types of synapses as well as at the neuromuscular junction - the specialized synapse that triggers the contraction of skeletal muscle.

The organophosphate compounds used as

insecticides (e.g., parathion) and

nerve gases (e.g. Sarin)

bind to the serine at the active site of acetylcholinesterase blocking its action.

Serpinlike Molecules

Angiotensinogen

This peptide is the precursor of angiotensin II - a major factor in maintaining blood pressure.

Chicken Ovalbumin

This is the major protein in the "white" of the egg (and a favorite antigen in immunological research).

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