Metal Dependent Dynamic Equilibrium: A Regulatory Mechanism for M17 Aminopeptidases from Plasmodium falciparum and Plasmodium vivax

Abstract

M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and Plasmodium vivax (Pv-M17) function as catalytically active hexamers to acquire free amino acids from human hemoglobin and are drug targets for the design of novel anti-malarial agents. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species, that can be controlled by manipulating the identity and concentration of metal ions available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of the Pv-M17 hexameric species revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of solution studies and structures determined by cryo-electron microscopy, we propose a dynamic equilibrium between monomer dimer tetramer hexamer, which becomes directional towards the large oligomeric states with the addition of metal ions. M17 aminopeptidases can exploit this sophisticated metal-dependent dynamic equilibrium to regulate formation of the catalytically active hexamer and therefore regulate catalysis.