Biomedical Engineering Reference
In-Depth Information
are targeted to a specific enzyme. Westling et al.
30
studied the substrate spe-
cificity of PfPMII by using a series of peptides with substitutions at different
positions along the length; this effort led to improved substrates for assay of
activity and inhibition. By examining the structure provided by Silva et al.
31
,
Westling and colleagues were able to identify Met 13, Ser 77, and Ile 287 as
potentially responsible for some of the aspects of specificity exhibited by the
enzyme. The Ser77Asp mutant gained the ability to cleave a substrate with a
Lys in the P1 position as do fungal enzymes with Asp in a similar position. The
Ile287Glu and Met13Glu/Ile287Glu mutant forms were able to process sub-
strates with positively charged amino acids in the P2 position.
Le Bonniec et al. found that PfPMII was active at neutral pH in the cleavage
of host erythrocyte spectrin, actin, and protein 4.1 from erythrocyte ghosts.
38
These results suggest that plasmepsin may play a second role in cleavage of
cytoskeletal elements of infected erythrocytes. As there are many events in
parasite invasion, growth, and exit from the human erythrocyte that are
undefined at this point, the experiments of this group may set the stage for
indentifying specific activities of the parasite enzyme.
When the parasite takes up hemoglobin and transports it to the digestive
vacuole, it also brings plasmepsin II along because the precursor of PfPMII is
anchored in the membrane of the transport vesicles through their long N-
terminal extensions.
39
At an appropriate point, it is the malarial cysteine
peptidases known as falcipains that cuts the mature plasmepsin from its
membrane anchor as demonstrated by the work of Drew et al.
40
The issue of
processing from the precursor to the mature enzyme was also studied by Kim
et al.
41
, who found evidence that the processing can be self-catalyzed in both cis
and trans processes (i.e., one molecule can cleave its own prosegment, or it can
cleave the prosegment of another molecule). However, it appears that these
studies were carried out in vitro using the truncated versions of the proenzymes;
in the native state, where the plasmepsins are membrane-bound, it is likely that
either the falcipains or calpains are involved in the conversion.
Specific interactions of substrate residues, as far out as the P5 position,
influence the eciency of cleavage of hemoglobin-related peptides.
42
This has
led to the suggestion by these authors that PMII may have the ability to induce
denaturation of hemoglobin at the point where cleavage is initiated.
11.3.3 HAP, the Histo-Aspartic Plasmepsin
The discovery of a gene with the substitution of a His for one of the catalytic
Asp residues
43
presented an exciting opportunity to perhaps design selective
inhibitors that could kill P. falciparum, which is the deadliest form of malaria.
Berry and colleagues showed that the gene was expressed in mid- and late
trophozoites as well as in young schizonts. They also provided preliminary
modeling of the new plasmepsin but did not report successful expression of the
protein. Bjelic and Aqvist reported
44
on the predicted structure, substrate
binding, and mechanism of action of HAP, and Popov et al. modeled substrate
and inhibitor binding of HAP.
45
