CHROMOGENIC SUBSTRATES UNIVERSITY | SERINE PROTEASES
What is a chromogenic Substrate?
Specificity and Selectivity
Substrates in Practice
Protein Concentrations in Plasma
Theoretical Basis for Calculation
International Units and Enzyme Activity
The most extensively studied group of proteolytic
enzymes comprises the serine proteases. As indicated by the name each member of
this group have a reactive seryl amino acid residue in its active site.
The serine proteases are divided into two families: the trypsins and the subtilisins.
The trypsin family is the largest and contains, among others, trypsin and chymotrypsin, elastase, mast cell tryptase, and many of the factors regulating blood coagulation and fibrinolysis.
The trypsin type of enzymes have a highly similar amino acid content. They are found in vertebrates and other animals, as well as in fungi and procaryotic cells. In contrast, the subtilisins are only found in bacteria. Members of the trypsin family are classified according to the type of amino acid that occurs at the preferred cleavage site.
Elastase and chymotrypsin cleave after hydrophobic and aromatic amino acids, while other trypsin-like proteases cleave only at the C-terminal side of the basic amino acids arginine or lysine. The amino acid sequence and thus also the three-dimensional structure differ completely between the trypsins and the subtilisins. The catalytically active domains of trypsin and subtilisin have therefore most probably evolved independently, converging from two different genes.
However, since the three amino acids of functional importance at the active sites, serine (Ser), aspartic acid (Asp) and histidine (His), are arranged in the same geometrical relationship in all members of the two families the proteolytic mechanisms are very similar.
This fact may lead to the suggestion that the arrangement of the three catalytically active amino acids at the active site is very efficient for hydrolysis of peptide bonds. Mammalian serine proteases are usually synthesized as inactive proenzymes, zymogens, consisting of a single peptide chain. Activation occurs when the zymogen is cleaved at one or several specific sites. Most commonly such cleavage is accomplished by the action of another protease. Most serine proteases contain two functionally distinct parts.
The region where the catalytically active amino acids are found is very similar in trypsin and chymotrypsin as well as in the serine proteases involved in blood coagulation. The other region is located in the exterior parts of the enzyme. This region is of considerable size in the serine proteases regulating blood coagulation and fibrinolysis and four main types of structures can be distinguished: kringle domains, growth factor domains, vitamin K dependent carboxylated calcium binding domains, and domains homologous to the finger structure of fibronectin.
All four domain types are not present in all groups of serine proteases.
In the living organism, proteolytic enzymes (proteases) are produced to degrade and modify proteins. A main task for proteolytic enzymes is to degrade proteins into peptides or amino acids to be used either as an energy source or as building blocks for resynthesis of proteins. Furthermore, proteolytic enzymes modify cellular environments and facilitate cell migration in connection with wound repair and cancer, ovulation and implantation of the fertilized egg, embryonic morphogenesis, and involution of mammary glands after lactation.
Another important function of the proteases is their role as regulators in processes such as inflammation, infection and blood clotting. Most proteolytic enzymes are highly specific for their substrates. The classification of proteases, however, is not based on their choice of substrate but on their mechanism of action.
Four different groups of proteolytic enzymes, named after the active site amino acid residue responsible for the catalytic activity, are generally distinguished: the aspartic proteases (e.g. pepsin), the cystein proteases (e.g. cathepsin B and cathepsin H), the serine proteases (e.g. trypsin, thrombin and plasmin) and metalloproteases (e.g. collagenases and gelatinases). Although the members of each group of proteolytic enzymes may have very diverse biological functions, amino acid analysis often shows a high degree of structural similarity between them. Detailed knowledge of the structure and mechanism of action of one enzyme can in many cases reveal an understanding of the structure and functions of other enzymes within the same group.
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