Biochemical Properties and Functional Applications of Proteinase K
Proteinase K (EC 3.4.21.64) is a highly active, broad-spectrum serine protease originally isolated from the fungus Engyodontium album (formerly Tritirachium album). It belongs to the subtilisin-like family of enzymes (subtilisins) and is named "K" because of its unique ability to digest keratin, a fibrous structural protein that is resistant to many other proteases.
Molecular Structure and Catalytic Mechanism
Proteinase K is a single-peptide chain protein with a molecular weight of approximately 28.9 kDa. Its catalytic activity is governed by a classic "catalytic triad" consisting of three specific amino acids: Aspartic Acid (Asp-39), Histidine (His-69), and Serine (Ser-224).
The enzyme functions by cleaving peptide bonds, specifically at the carboxylic sides of aliphatic, aromatic, or hydrophobic amino acids. Unlike many other enzymes that require specific conditions to maintain stability, Proteinase K is remarkably robust due to the presence of two disulfide bridges and two bound calcium ions, which protect the structural integrity of the protein even under harsh conditions.
Key Characteristics: Stability and Versatility
The primary reason Proteinase K is a staple in molecular biology laboratories is its extreme stability across a wide range of environments:
pH Range: It remains active across a broad pH spectrum, typically from pH 4.0 to 12.0, with an optimum near pH 8.0.
Temperature Stability: While it functions well at room temperature, its activity increases significantly at higher temperatures. It is commonly used at 55°C to 65°C, temperatures that would denature most other enzymes.
Resistance to Detergents: Remarkably, Proteinase K is stimulated by denaturing agents such as Sodium Dodecyl Sulfate (SDS) and urea. These agents unfold proteins, making the peptide bonds more accessible to the enzyme for faster digestion.
Chelating Agents: It remains active in the presence of EDTA, which is frequently used in DNA extraction buffers to inhibit DNases (enzymes that degrade DNA) by sequestering metal ions.