Biomedical Engineering Reference
In-Depth Information
In the binding of active site ligands to enzymes of this class, it is well established
that considerable movement of some parts of the protein occurs to lock down on
the bound inhibitor. This transition from the 'open' or free enzyme to the enzyme
in complexes with inhibitors was also seen
26
for plasmepsin I, with the flap region
of residues 70-83 moving to allow interaction with a bound ligand. However,
loops composed of residues 47-51, 106-114, 239-244, and 278a-282 also show
movement in the transition between the unbound and bound states.
One inhibitor used in by Bhaumik et al. was KNI-10006, one of a series of
peptidomimetic inhibitors synthesized in the laboratory of Prof. Yoshiaki Kiso
in Kyoto. In the plasmepsin I/KNI-10006 structure, it was observed that the
inhibitor bound in the opposite direction from that observed for other peptide-
like inhibitors in other aspartic proteinases including other plasmepsins
described below. The binding of other peptidomimietics was the same as that
expected for the binding of a substrate, when the directionality is defined from
the N-terminal to the C-terminal ends of the inhibitor or substrate. Placing the
P1 and P1
0
groups in the S
0
1 and S1 subsites of plasmepsin I, respectively,
provides the optimal binding for KNI-10006. A similar case will be described
for plasmepsin IV in a later section. Bhaumik et al. have also provided a table
28
that compares the amino acids involved in interactions at six different subsites,
S2 through S4
0
(although the residue in P3
0
does not interact with any enzyme
side chains). This information will be useful in designing specific inhibitors.
11.3.2 Plasmepsin II
The gene for plasmepsin II was found during sequencing studies of P. falciparum
in the laboratory of Professor Dame.
7
The 33% identity to the amino acid
sequence of human cathepsin D was noted early on, and this led to the cloning of
the full-length gene and expression studies. Following these initial discoveries,
it was found that the prosegment of proplasmepsin 2 could be truncated to
48 amino acids to yield high levels of expression and activity.
29
The folding
and self-activation of the truncated proplasmepsin II (denoted here as 'semi'-
proplasmepsin II) were optimized for the isolation of active material. Hill et al.'s
procedure
29
was used to prepare plasmepsin 2 (hereafter identified as PfPM2) for
a number of studies, including analysis of substrate specificity, inhibitor binding,
and X-ray crystallography. PfPM2 has proven to be a good target for drug
discovery, as many groups have used the enzyme to test a wide variety of com-
pounds, both naturally occurring and synthetic, as inhibitors. Some, but not all,
of these have been shown to kill the malaria parasite in culture, reinforcing the
conclusion that inhibitors of the aspartic proteinases are viable drug candidates.
A few words about plasmepsin II and its favorable properties are important.
It was found that the gene for semi-proplasmepsin II expressed well in E. coli,
yielding inclusion bodies that could be isolated and washed to remove
unwanted materials. This resulted in a preparation of the protein that was
nearly pure, as assessed by SDS-PAGE analysis.
30
Of course, at this stage, the
protein is in an almost completely unfolded and inactive state. Solubilizing the
inclusion bodies in 8 M urea and a pH of 8.0 gave a solution that could then be
