Biomedical Engineering Reference
In-Depth Information
dialyzed to yield a high percentage of correctly folded and potentially active
material. This is not always achieved from inclusion bodies for recombinant
proteins, thus marking plasmepsin II as a good choice for producing a
recombinant protein for further studies. In addition, the discovery of condi-
tions to affect the conversion to the mature enzyme (with a short N-terminal
extension), namely, incubation at pH 4.5 for 30 min, led to the production of
the active enzyme in good yield and purity. The best part is that the protein
converted to the mature form did not digest itself during the conversion,
probably due to the high level of correct folding, as natively folded proteins are
not subject to proteolytic digestion in most cases.
Silva et al.
31
reported the first structural analysis of plasmepsin II in complex
with pepstatin, the classic inhibitor of the aspartic peptidase family; in fact, this
was the first structure of any plasmepsin. The 33% identity with human cathepsin
D allowed these investigators to utilize the known structure of cathepsin D as the
model for molecular replacement. By using pepstatin as the active site ligand,
Silva and colleagues were able to make comparisons to other members of the
aspartic peptidase family where structures were solved with pepstatin bound. The
relatively small size of the side chains along the sequence of pepstatin is related to
the ability to bind to a variety of enzyme active sites. This paper also presented
information on the analysis of binding of several peptidomimetic inhibitors where
the phenylstatin moiety was utilized as a peptide bond replacement (Figure 11.2).
Figure 11.2
Comparison of the structure of plasmepsin II (PDB: 1SME, Silva et al.
31
)
and human cathepsin D (PDB: 1LYA, Baldwin et al.
118
). In this figure,
one can see that although there are small differences in the exact position
of the secondary structure elements (helices and beta-strands) these
appear at nearly identical locations in three dimensional space. Elements
of the structure, including the orthogonal beta sheets in each domain and
the six-stranded beta sheet at the bottom of each enzyme are very similar.
