Structural Biochemistry/Enzyme Catalytic Mechanism/Proteases

Proteases are enzymes that accelerate the hydrolysis of peptide bonds. In essence, proteases break up proteins into smaller peptide fragments. Proteases generally promote the hydrolysis of a peptide by activating a nucleophile, polarizing the peptide carbonyl and stabilizing the tetrahedral intermediate. Protease like all enzymes are very specific so recognizes side chain to know where to cleave.

Mechanism of Action
Proteases generally activate a nucleophile, which will in turn attack the carbon of the peptide bond. The electrons in the carbon-oxygen double bond migrate onto the oxygen as the nucleophile attaches itself. This tetrahedral intermediate is a high energy intermediate and the protease will generally have a way to stabilize this intermediate. The intermediate will then decompose, usually releasing the two peptide fragments.

The 4 main class of proteases are: Serine Proteases, Cysteine Proteases, Aspartyl Proteases, and Metalloproteases. All four classes of proteases utilize either use a different nucleophile or a different way to activate the nucleophile. Serine and cysteine proteases use a catalytic triad to activate the side chain of either a serine or cysteine. Aspartyl proteases use an aspartic acid residue to activate a water molecule and another aspartic acid residue to align the peptide for attack. Metalloproteases use a metal ion to activate a water molecule.

Stabilization of the tetrahedral intermediate is generally accomplished by parts of the proteins that aren't the active site. A common method is the use of an oxyanion hole. An oxyanion is a part of the protease which will encompass the tetrahedral intermediate. Within this hole, hydrogen bonding between the NH groups of the protease and the negatively charged oxygen of the protein will stabilize the intermediate.

Specificity of Proteases
An amazing feature of proteases is their preference for cleaving the peptide bond associated with a specific amino acid. This preference is a result of the active site's location within the protease's structure. The active site is generally in a cavity of the protein. The type of amino acid residues within the pocket will determine the preference of the proteases. Protease can also break ester bonds.

Chymotrypsin has a deep cavity made up of mostly hydrophobic residues, thus Chymotrypsin has a preference to cut peptide bonds of amino acids with large hydrophobic side chains such as tryptophan and phenylalanine. Elastase has bulky valine residues within the cavity, thus Elastase has a preference to cut peptide bonds of amino acids with small side chains. Trypsin has a aspartate residue, which has a negatively charged side chain, at the bottom on the cavity. Thus Trypsin has a preference to cut peptide bonds of amino acids with positively charged side chains.

Enzyme inhibition by DIPF: serine 195's Hydrogen can bind with Fluoride from DIPF and inhibit its nucleophilic attack on the carbonyl of peptide it wants to cleave.

Protease Inhibitors
The conversion of a zymogen to a protease through the cleavage of one peptide bond is an accurate way to switch on certain enzymatic activities. The following activation step is irreversible. Therefore a different mechanism is required to stop proteolysis with the help of specific protease inhibitors. There are several important drugs that serve as protease inhibitors. These inhibitors are specific for one enzyme and do not interfere in the production of other proteins in the body. For example, the inhibitor Indinvar is specific for the HIV protease because the interaction of water and the enzyme is not possible in other aspartyl proteases.

Captopril is an inhibitor for the metalloprotease antiotensin-converting enzyme (ACE). This inhibitor helps in the regulation of blood pressure in the body.

There are several HIV protease inhibitors used for the treatment of AIDS. The HIV protease is an aspartyl protease that cleaves multidomain viral proteins into their active forms. Indinavir is an inhibitor that structurally resembles peptide substrate of HIV protease by mimicking the tetrahedral intermediate. In the active site, indinavir adopts a conformation that is similar to the twofold symmetry of the enzyme. Two flexible flaps of the HIV protease's active site fold down on top of the bound inhibitor. The central alcohol interacts with the two aspartate residues of the active site. Plus, the inhibitor's two carbonyl groups are hydrogen bonded to a water molecule (this is not seen in the molecule below), which is also hydrogen bonded to a NH group in each of the flaps.



Enzymatic Trypsin Inhibitor
Another example of a protease inhibitor is known as Pancreatic Trypsin Inhibitor. It is a 6 kilodalton protein and inhibits trypsin by binding very strongly to the active site of trypsin. The trypsin and pancreatic trypsin inhibitor complex is very stable in that it has a dissociation constant of about 0.1 pM (standard free energy of -75 kJ/mol). THis means that the complex cannot be dissociated into its denatured state with common denaturing agents such as 8 M Urea or 6 M guanidine hydrochloric acid. Because pancreatic trypsin inhibitor is a very effective substrate, the complex that it forms with trypsin is extraordinarily stable. Also, analysis by X-rays reveal that the inhibitor lies in the active site in such a position that the lysine-15 side chain of the inhibitor interacts with an aspartate side chain of trypsin in the active site. Additionally, many hydrogen bonds between the main chain of trypsin and the inhibitor further stabilize the trypsin - pancreatic trypsin inhibitor complex.

After binding to the active site of trypsin, pancreatic trypsin inhibitor does not change its structure, which means that the inhibitor is preorganized into such a structure that is complementary to the enzyme's active site. This is seen by the slow rate of cleavage of the peptide bond between lysine-15 and alanine-16. Overall, this inhibitor is more like a substrate and its inherent structure is extremely complentary to the enzyme's active site that it binds really tightly and is turned over slowly.

The amount of physiologically available trypsin is greater than the amount of trypsin inhibitor. Since trypsin activates other zymnogens, inhibitors of trypsin needs to exist to prevent small amounts of trypsin from starting a mistakenly activated cascade.

α1-Antitrypsin (α1-antiproteinase)
α1-Antitrypsin is a 53 kilodalton plasma protein protease inhibitor. It protects tissues from digestion by elastase, which is a secretory product of white blood cells that engulf bacteria. α1-Antitrypsin inhibits elastase much better than it inhibits trypsin. Similar to pancreatic trypsin inhibitor, α1-Antitrypsin blocks the action to target enzymes by almost irreversibly binding to the enzyme active sites.

α1-Antitrypsin is a physiologically important inhibitor because without it, excess elastase destroys alveolar walls in the lungs by digesting connective-tissue proteins. This condition is called emphysema, in which people with this condition have difficulty breathing. People with emphysema must breathe harder than normal in order to exchange the same amount of oxygen as people without emphysema due their damaged alveoli. Cigarette smokers are more likely to develop emphysema because smoke oxidizes methionine-358 to methionine sulfoxide (see figure) of the α1-Antitrypsin inhibitor, which is an essential residue for binding elastase. The insertion of a single oxygen into the resulting methionine sulfoxide of the protein, which changes the inhibitor's affinity for elastase is a great example of the importance of structural biochemistry and the role it plays throughout physiological processes.

Reference
1. Berg, Jeremy; John L. Tymoczko, Lubert Stryer (2007). Biochemistry, 6th Edition. W. H. Freeman and Company, New York, New York.