A protease is a class of enzyme that breaks peptide bonds by hydrolysis and thus often functions to degrade protein. However, these enzymes have diverse functions that also include cell signalling, regulation of the cell cycle, antigen presentation in the immune response and the activation of proteins such as the hormone insulin by specific cleavage. They also play important roles in certain diseases such as emphysema, Alzheimer's Disease, cancer metastasis, HIV infection, and heart disease. They may also be referred to as peptidases or proteolytic enzymes.

                  H   H                    H   H
                   \ /                      \ /
                    N                        N
                    |                        |
                  H-C-R                    H-C-R
                    |                        |
                    C=O      + Water         C=O     H   H
                    |     -------------->    |        \ /
                 H- N                        OH   +    N
                    |                                  |
                  H-C-R'                             H-C-R'
                    |                                  |
                    C=O                                C=O
                    |                                  |
                    OH                                 OH

Hydrolysis of a single peptide bond. For simplicity a single dipeptide is shown. (R and R' represent any one of the 20 amino acid side chains).

A protease may degrade peptide bonds in something as small as a dipeptide or as large as a protein depending on the specificity of the enzyme's active site. The active site is the part of the enzyme that is responsible for the catalysis of a chemical reaction (hydrolysis in this case) and can be conveniently visualised as a cleft. Depending on the shape of this cleft different substrates will fit and be held in place in order for catalysis to occur. Schechter and Berger devised a simplified model for the active site of a protease in which the catalytic site is flanked by specificity subsites which can be thought of as pockets designed to fit the side chain of specific amino acids from the substrate, conventially numbered S1, S2, S3 etc. in the direction towards the N-terminus of the enzyme and S1', S2', S3' in the direction of the C-terminus. Similarly the amino acid residues in the protein/peptide substrate are numbered P1, P2, P3 etc. and P1', P2', P3' etc. depending on which specificity subsite each fits into. The size, shape and charge of a protease's specificity subsites will determine which amino acids will fit and what substrates will undergo peptide bond hydrolysis.

Examples of proteases include pepsin which helps degrade proteins in the stomach, trypsin which is secreted by the pancreas and also involved in protein digestion, thrombin which aids clot formation by conversion of soluble fibrinogen to insoluble fibrin, plasmin which helps dissolve blood clots and angiotensin-converting enzyme which forms the active hormone angiotensin II from angiotensin I to maintain blood pressure as part of homeostasis.

Classes of Protease

Proteases may be defined on the basis of where they cleave a peptide bond within their substrates and by the mechanism of their catalysis. For example, an endopeptidase cleaves peptide bonds within a peptide chain while an exopeptidase cleaves peptide bonds at the end of a peptide chain. If classified after the catalytic mechanism a protease is defined by the amino acid or co-factor involved. For example a serine protease uses a serine residue for nucleophilic attack of the peptide bond, aspartic proteases use an aspartate residue to cause an activated water molecule to act as a nucleophile and metalloproteases use a coordinated metal ion such as zinc to similarly induce a nucleophilic attack by water. Threonine and cysteine proteases also exist and use essentially the same mechanism as serine proteases.


In vivo the action of proteases can be regulated in a number of ways. Regulation may come in the form of protease inhibitors, rate of synthesis or rate of degradation. Many proteases are synthesised in an inactive form called a zymogen (a type of proenzyme) which has a distorted active site. Zymogens can themselves be activated by proteolytic cleavage to yield the active form of the enzyme.

In vitro inhibition of proteases can help determine the mechanism of catalysis. For example using metal chelating agents such as EDTA will remove metal ions from the enzyme's active site if it is a metallopeptidase but will leave other proteases unaffected. Similarly di-isopropyl fluorophosphate binds irreversibly to the serine at the active site of serine proteases to cause inhibition. Inhibition of proteases is also important experimentally to prevent the degradation of a protein one might be studying. In contrast proteases can be useful experimentally in order to break peptides down into smaller chunks for peptide sequencing or to remove fragments of proteins to see if they are crucial in function or subcellular targetting.

Inhibition of protease function provides the potential for the treatment for many diseases such as those mentioned at the beginning of this node. Most notable is the recent advance in treatment of hypertension by vasopeptidase inhibitors which inhibit two different metallopeptidases (angiotensin-converting enzyme and neutral endopeptidase) to prevent the conversion of angiotensin I to angiotensin II, which causes vasoconstriction to increase blood pressure, and to prevent the degradation of natriuretic peptides which help to produce vasodilation. In this manner high blood pressure can be controlled by administration of these protease inhibitors.


Hooper N M, Essays in Biochemistry Volume 38: Proteases in Biology and Medicine, Chapter 1 - Proteases: a Primer, Portland Press, 2002.

Voet D, Voet J G, Biochemistry (2nd edition), John Wiley & Sons, Inc., 1995.