Aspartate Proteases

Aspartate Proteases I

A review [Dunn, Chem. Rev., 2002, 102, 4431] listed nine families of aspartate proteases, including some 450 enzymes, all of which use a pair of aspartates, typically Asp32 and Asp215, as the catalytic residues.

Consider examples from the two most abundant families: A1, the pepsin-like, and A2, the retroviral proteases.

Pepsin is the most-studied of the A1 group. It is a digestive enzyme, but functions in the stomach, rather than the intestines.

Pepsin is stable only at acidic pH, about 3.5; it rapidly becomes inactive at pH above 7

The zymogen is stable at high pH, and rapidly cleaves itself to pepsin as the pH is lowered

It is an endopeptidase, acting on the N-terminal side of amino acids with aromatic side chains, like chymotrypsin

Human Pepsin (1qrp) The Catalytic Site
- Like many other proteases, pepsin consists of two large domains with the catalytic site between them, formed by residues from each domain.
- The binding site is open enough to contain as many as six or seven residues surrounding the site of cleavage.
- The Ser and Thr residues shown in the catalytic center are believed by some to participate in the chemistry by hydrogen bonding to the aspartates
Porcine pepsin is very similar, aligning with the human enzyme to an rmsd for backbone atoms of 0.71 A:

Porcine Pepsin (3pep) Aligned with Human (1qrp)
(catalytic residues shown CPK)

A sequence alignment gives 84% identity of residues:

Sequence Alignment of Porcine (3pep) and Human (1qrp) Pepsin

Two slightly different mechanisms have been suggested for catalysis. In the first, the two aspartate carboxyls are hydrogen-bonded to a proton, forming a structure similar to the well known carboxylic acid dimer structure found in non-polar environments:
- The hydrogen bond is one of those "low-barrier" ones we have mentioned before
- The "two carboxylate - one proton" configuration is suggested by a bell curve for activity vs pH
- As suggested for zinc proteases, water is the nucleophile, with its attack facilitated by an aspartate
- The usual tetrahedral intermediate is formed
The alternative mechanism eliminates the "low-barrier" hydrogen bond, and substitutes hydrogen bonds to neighboring serine and threonine residues:
- Fundamentally, the distinction is the position of a proton: between the aspartates or attached to one of them
- Neutron diffraction on a crystal soaked in D₂O seems to favor the second mechanism, suggesting that only one of the aspartates is protonated
This page last modified 12:41 PM on Thursday March 24th, 2011.
Webmaster, Department of Chemistry, University of Maine, Orono, ME 04469