Peptide bond cleavage by PepA and leucine aminopeptidase

Biological background
E. coli aminopeptidase A (PepA) und bovine lens leucine aminopeptidase (LAP) share ~30% sequence identity. As the name implies, these exopeptidases cleave the N-terminal amino acids from peptides. Human LAP has been shown to catalyze postproteasomal trimming of the N-terminus of antigenic peptides for presentation on MHC class I molecules. Here, interferon-gamma not only promotes proteasomal cleavage but also indices LAP for N-terminal processing of the peptides.
In addition to the aminopeptidase activity, PepA (but not LAP) has independent DNA-binding functions.
 
 

Hexamer structure

E. coli aminopeptidase A (PepA) is a hexameric protein of symmetry 32, i.e. two-fold molecular axes are perpendicular to a three-fold molecular axis. In the figure on the left, the view is along the three-fold axis. The C-terminal domains are shown in blue and the N-terminal domains in green. A long helix (orange) connects the two domains. The catalytic zinc ions are shown in yellow.

Compartimentalization

The aminopeptidase active sites (marked by the catalytic zinc ions shown in red) are located in the center of the hexamer, where a large cavity of 30 Angstrom diameter and 10 Angstrom height is formed. Access to this cavity is provided by channels. Such a compartimentalization of the reaction room also occurs in other proteases and in the proteasome (see review by Larsen and Finley, 1997). In PepA and LAP the compartimentalization ensures that the enzyme acts only on small peptides (~6 residues) and not on proteins. In the left figure a LAP hexamer has been cut open perpendicular to the threefold axis. The cut protein regions are coloured green and the rest of the protein surface in blue and white. The active sites are marked in red.

The aminopeptidase active site

The electron density map of LAP at 1.6 Angstrom resolution reveals many details of the active site. A surprising finding was the binding of a carbonate ion next to an arginine residue. The structure of PepA, which was determined later, and kinetic studies on wild-type PepA and mutant variants showed that this carbonate ion is not an artifact of the crystallization conditions, but is part of the active site and it has a functional role. PepA is activated ~8-fold by physiological concentrations of bicarbonate ions, i.e. that are present in the cell from dissolved carbon dioxide.

In the unliganded structure both zinc ions are five-coordinated, mainly by oxygen atoms from carboxylate side chains, a peptide carbonyl group and a water molecule. A somewhat unusual metal ligand is the lysine residues coordinated to Zn2.

Inhibitor binding

Details on the enzyme mechanism were obtained from the binding of transition-state analogues to the catalytic center. Shown on the left is the binding mode of leucinal, in which the carboxylate group of leucine is replaced by an aldehyde group. The aldehyde group is hydrated to a gem-diol, which mimicks the gem-diolate group of the transition-state of the reaction. Both hydroxyl groups of the gem-diol are coordinated to the zinc ions. In addition, the amino group of the inhibitor is also bound to one of the zinc ions.  In the transition-state both zinc ions are six-coordinated.

Catalytic mechanism

References
Larsen, C. N. & Finley, D. (1997). Protein translocation channels in the proteasome and other proteases. Cell 91, 431-434.

Sträter, N., Sun, L., Kantrowitz, E. N.  & Lipscomb, W. N. (1999). A carbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase. Proc. Natl. Acad. Sci. USA 96, 11151-11155.

Sträter, N. & Lipscomb, W. N. (1995). Two-metal ion mechanism of bovine lens leucine aminopeptidase: active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, by X-ray crystallography. Biochemistry 34, 14792-14800.

Sträter, N. & Lipscomb, W. N. (1995). Transition state analogue L-leucinephosphonic acid bound to bovine lens leucine aminopeptidase: X-ray structure at 1.65 Å resolution in a new crystal form. Biochemistry34, 9200-9210.