Biomedical Engineering Reference
In-Depth Information
a housekeeping role in the removal of the initiator methionine from newly
synthesized polypeptides; two prolyl aminopeptidases, an aspartyl amino-
peptidase (PfA-M18); two neutral aminopeptidases (leucine aminopeptidase
PfA-M17 and alanine aminopeptidase PfA-M1), and an aminopeptidase P.
Given the restricted specificities each of these enzymes for particular N-terminal
amino acids, it is thought that they act in concert to facilitate protein turnover.
7.1.3 Pf Neutral Aminopeptidases as New Drug Targets
The absolute requirement of parasites to use MAP activity to digest host
hemoglobin makes this process ideal as a target for chemotherapeutic inter-
vention strategies, as there appears to be limited functional redundancy
between these enzymes. We, and others,
21-25
have shown that the generic
aminopeptidase inhibitor bestatin, an antibiotic and natural analog of the
dipeptide Phe-Leu derived from the fungus Streptomyces olivoretticul, prevents
Pf malaria growth in culture. Inhibition of the MAP activity within the parasite
blocks the hemoglobin degradation and depletes the pool of available free
amino acids, resulting in the blockade of protein synthesis and subsequently
halting parasite development.
25
Interestingly, while inhibitors of the DV
aspartic proteases and cysteine protease exhibited strong synergism in the
killing of malaria parasites in culture, inhibitors of the neutral aminopeptidase
(bestatin) have shown very low synergism with inhibitors of either of the former
proteases classes, indicating that they may function in the same general path-
way of hemoglobin digestion but in separate compartments of the cell.
23
Early studies suggested that MAP activities were absent from the DV,
leading to the proposal that the final steps of hemoglobin digestion occurred
within the cytosol of the parasite.
26,27
MAP activity was optimum at neutral pH
and inactive below pH 6.0, and showed a preference for synthetic substrates
containing leucine and alanine at the N-terminus.
27,28
These biochemical
properties were consistent with a cytosolic enzymatic function, and the pro-
posed role in the terminal stages of hemoglobin catabolism, since leucine and
alanine constitute approximately 24% of hemoglobin amino acids.
28
These
early data implied that neutral metallo-aminopeptidases were the predominant
enzyme type responsible for this activity.
27,28
Subsequent characterization of purified enzyme from parasites and recom-
binant production revealed that PfA-M1 and PfA-M17 are the only neutral
aminopeptidases in malaria cells. Both aminopeptidases have a strong pre-
ference for a P1 leucine residue.
26,29-31
An explanation for this apparent
redundancy is that besides regulating the general intracellular pool of amino
acids, PfA-M1 and PfA-M17 are critical in the hydrolysis of dipeptides that
release free leucine. The only amino acid not found in human hemoglobin and
which cannot be synthesized by the parasite is isoleucine. This amino acid must
be obtained from the external environment.
32
The uptake of isoleucine by
parasites is mediated by a two-step process involving an ATP-independent
transporter capable of exchanging intra-cellular leucine for isoleucine, and the
ATP-dependent storage or accumulation of this amino acid within parasites.
33
