Proteases are important reagents in biotechnology and molecular biology laboratories. The use of proteases is often necessary to inactivate protein contaminants in our prepared samples.
Proteases are enzymes that catalyze the cleavage of a protein into amino acids. These enzymes digest a protein by using an amino residue or an activated water molecule as a nucleophile. A nucleophile is an atom of an amino acid that donates an electron to another molecule, causing changes in another amino acid.
Proteases use two types of mechanisms to break a peptide bond:
1. The first mechanism for peptide bond breaking involves the use of an activated water molecule to perform a nucleophilic attack. Examples for these enzymes are aspartic-, glutamic, and metallo proteases.
2. The second mechanism involves the use of a nucleophilic residue to cleave a peptide bond. Serine proteases use an active serine to perform a nucleophilic attack. Cysteine peptidases use the sulfur in cysteine to perform nucleophilic attack. Threonine proteases use a threonine residue as a nucleophile.
The Classes of Proteases
Proteases can be divided into different classes based on the location of their cut, chemical structures, or catalytic mechanism.
Based on the cleavage site, proteases can be classified into:
- Endopeptidases: these enzymes cut internal peptide bonds.
- Exopeptidases: their catalytic activity is near the amino- or carboxy-termini of their substrates.
Different Classes of Peptidases. The endopeptidase breaks internal peptide bonds. The aminopeptidase cuts at the amino terminus of a substrate, whereas carboxypeptidase cleaves near a carboxy terminus of a substrate.
However, there is another way to classify proteases. Based on their chemical structures and catalytic mechanisms, there are six classes of proteases:
- Aspartic proteases: using an aspartatic acid in their catalytic mechanism.
- Glutamic proteases: using a glutamatic acid in their mechanism.
- Metalloproteases: using a metal ion to catalyze the reaction.
- Cysteine proteases: using a cysteine as a nucleophile.
- Threonine proteases : using a threonine in the catalytic mechanism.
- Asparagine peptide lyases: using an asparagine for an elimination reaction.
- Serine proteases: using a serine as a nucleophile.
Serine proteases are proteases characterized by the presence of a reactive serine residue in a catalytic triad. A catalytic triad is a group of three amino acids at the enzyme active site that are involved in catalysis. The catalytic triad allows the transfer of protons into and out of the active site. An example of a catalytic triad for chymotrypsin: Ser 195, His 57, and Asp 102. In this example, the three-letter words represent the amino acid, and the number represents their position.
In these serine proteases, the catalytic triad usually consists of serine, aspartic acid, and histidine amino acids. Some other groups of serine proteases may have a different combination of residues in the catalytic triad, such as Ser-His-Glu, Ser-Lys/His, or His-Ser-His. However, the homology studies and analyses of the active site among serine proteases show that all of these enzymes have a nucleophile serine and the same catalytic mechanism.
The catalytic mechanism of the serine protease chymotrypsin usually represents the catalytic mechanism for all of serine proteases. It is because the structure and mechanism of chymotrypsin is well characterized. Chymotrypsin catalyzes the cleavage of peptide bonds of proteins near the carboxyl group of the aromatic amino acid. The catalytic triad of chymotrypsin consists of Ser 195, His 57, and Asp 102.
The step-wise catalytic mechanism of chymotrypsin:
1. The active Serine (Ser 195) performs the nucleophilic attack on the carbonyl carbon atom of the peptide.
2. The nucleophilic attack causes the formation of tetrahedral intermediate.
3. The tetrahedral intermediate changes into an acyl-enzyme intermediate. The solvent water comes to replace the first amine product.
4. The first amine product leaves and the solvent water replaces it.
5. The reaction produces a second tetrahedral intermediate.
6. The final reaction causes the release of the second amine product and the formation of the active enzyme.
Serine protease consists of large class of proteases based on their structural similarity and functions. Below are some well-characterized groups of serine proteases:
- Chymotrypsin: Chymotrypsin has a specificity pocket with hydrophobic amino acid, therefore it attracts substrate proteins with hydrophobic amino acids. A specificity pocket is a pocket at the enzyme active site allowing a substrate to bind for cleavage.
- Trypsin: Trypsin has a negatively charged aspartic acid in the pocket, attracting a positively charged amino acid.
- Elastase: Members of elastase group have a small specificity pocket, so only a small amino acid enters it.
- Subtilisin: Members of this group show high specificity for aromatic and hydrophobic amino acids. Although chymotrypsin and subtilisin have different primary and tertiary structures, the catalytic triad of subtilisin resembles that of chymotrypsin.
What is Proteinase K?
Proteinase K is an endopeptidase belonging to the subtilisin group of serine proteases. In molecular biology laboratories, proteinase K is useful during preparation of DNA or RNA samples by degrading and inactivating proteins.
Proteinase K. Proteinase K was isolated from the mold (Parengyodontium album or previously known as Tritirachium album). Proteinase K’s name came from its catalytic activity using keratin as a substrate. The proteinase K model is created with SWISS-MODEL (https://swissmodel.expasy.org).
Proteinase K has five cysteines. Four of the cysteine residues form two disulfide bonds (34-124 and 179-248, respectively) and one lies below one of the catalytic triads. Proteinase K often binds two calcium ions required for full enzymatic activity.
Chemical Structure of Proteinase K
Proteinase K has the catalytic triad consisting of Ser 224, His 69, and Asp 39. The substrate recognition sites are two peptide chains, 99-104 and 132-136. It also has a free Cys 73 near His 69.
Catalytic Triad of Proteinase K from P. album. Catalytic triad model is created with SWISS-MODEL (https://swissmodel.expasy.org).
Why is Proteinase K Used for Mammalian DNA Isolation?
After the addition of a detergent, all sorts of material is released into lysate including harmful nucleases. To protect DNA from degradation, a protease, in this case proteinase K is used. The reason proteinase K is a good choice is because it’s pretty stable at higher temperatures and it’s broad-spectrum in terms of its targets.
How is Proteinase K Used for Mammalian DNA Isolation?
The first step for DNA isolation from mammalian cells and leucocytes is lysing the cells and spinning them down to collect a pellet. Once in suspension, detergent (SDS) is added to disrupt the nuclear membrane. Both histone and non-histone proteins release the DNA after digestion of protein by proteinase K.In addition, proteinase K digests and inactivates ribonuclease in the solution.
After digestion by proteinase K, phenol extractions separate DNA from the lipids and proteins. Hydrophobic lipids stay in the organic phase, whereas the DNA resides in the aqueous phase. Protein (including proteinase K) stays at the organic/aqueous interphase. DNA is extracted from the aqueous layer.Phenol extractions are important to eliminate proteinase K from the high molecular weight DNA.
Is Proteinase K Only Used for Mammalian DNA Isolation?
Proteinase K is useful during DNA isolation from other organisms as well, including:
Can Proteinase K be Used for RNA Isolation?
During RNA isolation, the addition of proteinase K degrades and inactivates even tiny amounts of ribonuclease in the presence of substrate. The additional phenol chloroform and isopropanol precipitation step reduces protein contamination and makes the final pellet easier to re-dissolve.
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