The M17 Leucine Aminopeptidase of the Malaria Parasite Plasmodium falciparum: Importance of Active Site Metal Ions in the Binding of Substrates and Inhibitors

Abstract

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

“…Our biochemical studies suggest that the ability of Pf A-M17 to rapidly gain or lose a divalent cation, most likely at the regulatory site 1, may represent an important mechanism of regulation of catalytic activity not only for the binding of substrates but also inhibitors (13). Consistent with this idea, we found that in the absence of environmental metal, no metal ion was observed at the catalytic site 1 position (SI Text Fig.…”

“…In contrast, the second metal ion is "tightbinding" and is known as the catalytic site 2 (18). Metal replacement studies on Pf A-M17 show that the enzyme retains activity when only the tight-binding catalytic site 2 is occupied with a metal ion; removal of both metal ions lead to complete and irreversible inactivity (7,13). Consistent with these ideas, in the presence of increasing concentrations of EDTA, loss of M17 activity follows a biphasic pattern as each metal ion is sequentially chelated (13) (SI Text Fig.…”

“…Pf A-M1 binds a single, tightly-bound Zn 2þ metal ion whereas Pf A-M17 contains two metal-binding sites, a readily exchangeable site (site 1) and a tight-binding site (site 2) (13). Whereas Pf A-M17 retains residual catalytic activity when the metal ion from site 1 is removed, the removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by addition of divalent metal cations (13). However, the divalent metal cation binding at site 1 of Pf A-M17 can be functionally exchanged for other metal cations, the enzyme displaying a preference in the order Zn 2þ > Mn 2þ > Co 2þ > Mg 2þ (13).…”

“…Whereas Pf A-M17 retains residual catalytic activity when the metal ion from site 1 is removed, the removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by addition of divalent metal cations (13). However, the divalent metal cation binding at site 1 of Pf A-M17 can be functionally exchanged for other metal cations, the enzyme displaying a preference in the order Zn 2þ > Mn 2þ > Co 2þ > Mg 2þ (13). The type of metal cation in the active site of these enzymes can therefore influence the catalytic efficiency against peptide substrates as well as the binding of inhibitors.…”

Metal ions affect insulin-degrading enzyme activity

“…Modulation of the proteolytic activity of IDE could therefore occur through different routes such as coordination of exogenous ligands to the catalytic metal, substitution or removal of the latter [44][45][46][47][48][49][50][51]. Moreover, metals could bind either to residues present in the active site to block substrate interaction or to residues outside the active-site to affect the structural integrity of the enzyme [42,43]; iii.…”