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Aspartic Proteases and Regulators

Introduction of Aspartic Proteases

There is a class of proteolytic enzymes of the pepsin family namely aspartic proteases (EC3.4.23). These proteases which always function in acidic solution share the identical catalytic domain. That's why aspartic proteases usually function to the specific locations in various organisms and they show less abundant than other groups of proteases, such as serine proteases. There sources of aspartic proteases are various, such as stomach (pepsin, gastricsin, and chymosin), lysosomes (cathepsins D and E), kidney (renin), yeast granules, and fungi (secreted proteases e.g. rhizopuspepsin, penicillopepsin, and endothiapepsin). Researchers have determined the amino acid sequences of some aspartic proteases. They find that although the biological sources and functions of aspartic proteases may be varying, their sequences are homologous and highly conserved. This may be due to the large substrate binding cleft and this structure can work to different substrates without additional domains, just as the case of serine proteases evolution. However, there are also some aspartic proteases with larger molecular weights (e.g. cathepsin E and a pituitary enzyme involving in the prohormone processing). More researches should be done to check if there are additional domains in these enzymes.

Classification of Aspartic Proteases

We can find the nomenclature of all these proteases in the MEROPS database and they are generally classified as cysteine, metallo-, serine, threonine and aspartic. The aspartic proteases (E.C.3.4.23) are also called acid proteases which are found in animals, fungi, plants, protozoa, bacteria and viruses. Those secreted by several microorganisms are called secreted aspartic proteases (SAPs) which have critical functions related to nutrition and pathogenesis.

Besides, according to their primary and tertiary structures, Barrett et al. classified these enzymes into families and clans (Figure 1). In one family the enzymes share a statistically significant relationship between their amino acid sequences in the active site. Each clan is a set of families in which their enzymes have a non-comparable primary structure after evolution although they once have the same ancestral protein. If these structures are inapplicable, people will consider the order of the residues in the active site of the polypeptide chain and the motif sequence next to the catalytic site as classification standard.

Families and clans of aspartic proteases

Figure 1. Families and clans of aspartic proteases

For example, as in the human genome there are only 15 members of aspartic proteases. They can be classified into two clans (clan AA and clan AD) based on their different tertiary structures. Family A1 of clan AA consists of the classical aspartic proteases (renin, pepsin A, pepsin C, cathepsin D, cathepsin E, BACE1, BACE2, and napsin A), and family A2 includes proteases integrated by retroviruses into the human genome such as HIV protease. In addition the presenilins and signal peptide peptidase as intramembrane cleaving proteases is belongs to the clan AD. The potential for these proteases as an important source of drug targets for the pharmaceutical industry is big despite the small member size.

Catalytic mechanism

Researchers have identified the crystal structures of most A1 human aspartic proteases. There are 3 topologically distinct regions namely an N-terminal domain, a C-terminal domain and a six-stranded anti-parallel β-sheet interdomain connecting the two other domains all of which will fold as a unit. Both, the N- and C-terminal domain contribute one catalytic aspartic acid residue to the active site respectively. Most clan AA aspartic proteases have a flap that closes down on top of the substrate or inhibitor, shielding the active site from solvent and forming the active site with binding pockets on both sides of the catalytic residues. Peptide bond cleavage occurs by a general acid-base catalytic mechanism (Figure 2). One of the two catalytic aspartic residues is protonated in the enzyme substrate complex. The other aspartic residue acts as a general base activating a water molecule which then attacks the carbonyl carbon of the scissile amide bond resulting in the formation of a tetrahedral geminal diol intermediate. Subsequent deprotonation of the hydroxyl group by one of the catalytic aspartates and simultaneous activation of the leaving amine by the other, protonated, aspartic residue ultimately leads to peptide bond cleavage.

Mechanism of amide bond hydrolysis by aspartic proteases. Catalytic aspartic residues are shown in red. P1, substrate residue N-terminal of the scissile bond binding in the non-primed side; P1', substrate residue C-terminal of the scissile bond binding in the primed side of the protease.

Figure 2. Mechanism of amide bond hydrolysis by aspartic proteases. Catalytic aspartic residues are shown in red. P1, substrate residue N-terminal of the scissile bond binding in the non-primed side; P1', substrate residue C-terminal of the scissile bond binding in the primed side of the protease.

Regulators of Aspartic Proteases

Although pepstatin produced by Streptomyces is an inhibitor of a wide spectrum of aspartic proteases. People find that SAPs are insensitive to pepstatin but sensitive to diazoacetyl-dl-norleucine methyl ester (DAN) and 1, 2-epoxy-3-(pnitrophenoxy) propane (EPNP) in the presence of copper ions. The aspartic protease inhibitors of SAPs are grouped as follows: Kunitz-type inhibitors, which are widely distributed in plants such as legumes, cereals and solanaceae; Ascaris inhibitors, which are isolated from the hemolymph of Apis mellifera; and the IA3 inhibitor, which is produced by the yeast Saccharomyces cerevisiae. Basically, researchers divide inhibitors into different categories by taking advantage of their molecular nature as protein inhibitors or low molecular weight inhibitors. Recently, there are more new generation aspartic protease inhibitors identified.

HIV- human immunodeficiency virus is the cause of the acquired immunodeficiency syndrome (AIDS), which is proved in the early 1980s. Then researchers find that HIV protease takes the responsibility of the cleavage of the viral poly-protein, and this is a critical step during the viral life cycle at the late stage. If we destroy the proteolytic activity of HIV protease, the viral particles of HIV will be immature and noninfectious. To date there are nine HIV protease inhibitor drugs approved, which can be used in clinical (i.e. Saquinavir, Nelfinavir, Ritonavir, Lopinavir, Indinavir, Amprenavir, Fosamprenavir, Atazanavir, and Tipranavir).

BACE-1 (β-amyloid precursor protein cleaving enzyme-1), also known as Asp2 and memapsin-2, is expressed in the brain and other tissues and it is a kind of membrane bound aspartic protease. The amyloid precursor protein (APP) will be proteolytic truncated to C99 when BACE-1 is activated in the brain, then generating amyloidogenic peptides Aβ40 and Aβ42 with the help of presenilins. The Aβ40 peptides are the major components of amyloid plaques accumulating in the brain of Alzheimer’s disease (AD) patients. A unique feature of the BACE-1 structure is the loop defined as the S1’/S3’ pocket. The loop of cathepsin D and renin is small and will be truncated to allow inhibitors to cover a larger space with more hydrophilic character. On the contrary the S1/S3 pocket of BACE-1 is large and hydrophobic. Using this characteristic, researchers are trying to design inhibitors of BACE-1 to discovery the drugs for the treatment of Alzheimer’s disease.


  1. Eder J, et al. Aspartic proteases in drug discovery. Current Pharmaceutical Design. 2007, 13(3).
  2. Tang J, Wong R N. Evolution in the structure and function of aspartic proteases. Journal of Cellular Biochemistry. 2010, 33(1).
  3. Mandujano-Gonzà lez V, et al. Secreted fungal aspartic proteases: A review. Revista Iberoamericana De Micologia. 2016, 33(2):76-82.
  4. Mello L V, et al. Identification of novel aspartic proteases from Strongyloides ratti and characterisation of their evolutionary relationships, stage-specific expression and molecular structure. Bmc Genomics. 2009, 10(1):611.

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