Proteolytic enzymes are grouped into families and clans based on sequence relatedness. They are classified by their substrate specificity or by their mechanism of enzyme action. In many situations that involve protein degradation, proteases of diverse mechanistic classes and substrate specificities work in concert to degrade protein substrates. To illustrate, it has been suggested that the proteasome breakdown of cytoplasmic proteins in response to toxicity by heavy metals is continued by a combination of cytosolic endopeptidases and an exopeptidase. The endopeptidases described were tripeptidyl peptidase II (TPPII), a subtilisin-type protease and thimet oligopeptidase (TOP), a zinc-dependent cytosolic metalloproteasase; while the exopeptidase was leucyl aminopeptidases (LAP), also a zinc-dependent metalloprotease. Before arriving at understanding of cooperative action of the proteases in processing or bulk degradation of specific protein substrates, it is instructive to understand how individual proteins are grouped and the rationale behind such groupings.
The human genome encodes >550 proteases, which are divided into five classes on the basis of catalytic mechanism. Cysteine, serine, and threonine proteases use an amino acid residue in the enzyme active site (Cys, Ser, or Thr, respectively) as a nucleophile to attack the scissile amide bond of the peptide or protein substrate that is to be hydrolyzed.
As genome sequences have become available, it is common to see lists of gene families. MEROPS, the peptidase database, lists genes coding for proteolytic enzymes or protease-like molecules.
Proteases can be classified according to mechanism of action. They are referred to by the type of amino acid residue (or metal cation) that is most critical for their catalytic activity. Using this mechanistic classification, the main classes of independently acting proteases include the serine, cysteine, aspartic, and metallo-proteases.
Serine proteases are characterized by the presence of a serine residue working with a histidine and an aspartic acid residue in the catalytic triad. Proteolysis is initiated by nucleophilic attack at the carbonyl carbon of the peptide bond. Nucleophilic attack is by the serine hydroxyl that is made more nucleophilic by the negatively charged Asp working through the imidazole group of the histidine.
Cysteine (sulfhydryl) proteases are characterized by the presence of a nucleophilic cysteine thiol in the active site of the enzyme. The catalytic mechanism is very similar to that of serine proteases except that the nucleophilic attack is by sulfhydryl group of a Cys residue at the active site.
Aspartic proteases have two aspartic acid residues at the active site. One Asp residue acts as a base to make the oxygen of water more nucleophilic allowing for the attack of the carbonyl carbon of the peptide bond to be hydrolyzed. The other Asp residue provides a proton for the new N-terminus of the cleaved polypeptide chain.
Metalloproteases comprise a very diverse group of endopeptidases and exopeptidases. All are characterized by the presence of a catalytic divalent metal ion in the active site, usually zinc, sometimes cobalt, nickel, or manganese. At the catalytic site, the imidazole group of two histdine (or lysine, aspartate, glutamate) residues serve as ligands to the divalent metal cation. Similar to aspartyl proteases, metalloproteases activate a water molecule to serve as the nucleophile that attacks the carbonyl carbon of the scissile peptide bond.
Within each mechanistic class, proteases are further classified into families and clans based upon their amino acid sequence and evolutionary relatedness. To illustrate with examples that are generally familiar, there are three superfamilies of serine proteases. The best-studied members of the trypsin family are the ones that function in mammalian digestion of food proteins. Because they are well studied and are easily available commercially in pure form, these enzymes are often used to establish new methods, as will be shown in this dissertation. The ones we use are trypsin, chymotrypsin and elastase. All three are endopeptidases with no restriction with regard to the nature of the protein substrate, but with specificity for peptide bonds hydrolyzed. Trypsins hydrolyze peptide bonds where the carbonyl group is that of Lys or Arg; chymotrypsin prefers peptide bonds where the carbonyl group belongs to a large hydrophobic amino acid such as tyrosine, tryptophan, and phenylalanine while elastase is specific for those where the carbonyl group is that of a small residue such as Gly or Ala. The enzymes within the trypsin/chymotrypsin superfamily have similar sequences and three-dimensional structures, and are thought to be evolutionarily related to a common ancestor. Members of one superfamily do not share sequence homology with members of other superfamilies; having evolved independently from different ancestral genes.
The pH optima for proteolytic enzymes span the entire range so that some are also classified as acidic, neutral or alkaline proteases (although sometimes these terms are used in reference to the pI of the enzyme). The pH optimum may reflect subcellular localization. For instance, enzymes in the vacuoles, the apoplast and the thylakoid lumen generally have acidic pH optima whereas proteases in the cytosol, mitochondrial matrix and chloroplast stroma will in general be neutral or alkaline. Therefore, knowing the pH optimum gives a general indication of the subcellular localization
Proteolytic enzymes reference
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