Biomedical Engineering Reference
In-Depth Information
Banerjee et al. showed that there were four different plasmepsins in the food
vacuole; HAP, histoaspartic proteinase, and plasmepsin IV were the two
additional ones.
17
In HAP, one of the two conserved aspartic acids is replaced
by a histidine to give an unusual enzyme. This will be discussed further below.
Of the remaining six plasmepsin genes within P. falciparum, three (plas-
mepsins VI, VII, and VIII) are not expressed at high levels in the erythrocytic
stage of infection. Most likely they are either expressed in the liver stage of the
life cycle or in the sexual stage in the mosquito host. As current inhibitor design
strategies focus on the blood-borne stage, our attention will be focused on
plasmepsin I, II, HAP, and IV in the food vacuole and V, IX, and X in other
compartments in the parasite.
11.3 Digestive Vacuole Enzymes
As described above, initial efforts were focused on the possibility of finding
compounds that would block degradation of hemoglobin in the digestive
vacuole. It was known that approximately 75% of the hemoglobin protein in a
red blood cell was degraded within the digestive vacuole during the growth of a
parasite. It has been shown that the pH of the digestive vacuole is 5.4-5.5 by
using a GFP-PfPMII fusion protein.
18
Thus, it was quite reasonable to think
that this was an essential process to provide amino acids for the synthesis of
new proteins for the growing parasite and that aspartic peptidases were
involved. In addition, the period of the 1990s was dominated by studies of
plasmepsins I and II because these were the first enzymes expressed and pur-
ified. The following paragraphs will present information on each of these
enzymes in a sequential way, but not necessarily in a chronological fashion.
11.3.1 Plasmepsin I
Goldberg and colleagues described the cloning of plasmepsin I and some of its
functional properties.
4,6,19,20
Moon et al. described studies of the expression and
partial purification of plasmepsin I.
21
Subsequently, Tyas et al. compared native
and recombinant plasmepsin I and found that the recombinant protein, which was
extended by 12 residues at its N-terminus, gave a three- to 10-fold reduction in
catalytic parameters but identical K
i
values for several inhibitors.
22
Siripurkpong
et al. compared the active site residues of plasmepsins I and II, and attempted
mutagenesis to determine the cause of differences in specificity.
23
Xiao et al. uti-
lized a novel thioredoxin fusion approach to make plasmepsin I and reported on
the resulting properties of the product.
24
They found that their product exhibited
comparable kinetic properties to the native enzyme. Liu et al. made a mutation
within the prosegment of plasmepsin I and produced a higher yield of the mature
enzyme
25
than had been reported by Moon et al. This preparation was utilized for
studies of the substrate specificity of plasmepsin I, and inhibitors were designed
based on the resulting data. This will be discussed further in a later section.
26
Xiao et al.
27
created an expression construct with a truncated version of pro-
plasmpesin I following a thioredoxin fusion segment and tags for identification of
