Engagement of the S1, S1′ and S2′ subsites drives efficient catalysis of peptide bond hydrolysis by the M1-family aminopeptidase from Plasmodium falciparum

Abstract

The M1-family aminopeptidase PfA-M1 catalyzes the last step in the catabolism of human hemoglobin to amino acids in the Plasmodium falciparum food vacuole. In this study, the structural features of the substrate that promote efficient PfA-M1-catalyzed peptide bond hydrolysis were analyzed. X-Ala and Ala-X dipeptide substrates were employed to characterize the specificities of the enzyme's S1 and S1’ subsites. Both subsites exhibited a preference for basic and hydrophobic sidechains over polar and acidic sidechains. The relative specificity of the S1 subsite was similar over the pH range 5.5 - 7.5. Substrate P1 and P1’ residues affected both Km and kcat, revealing that sidechain-subsite interactions not only drive the formation of the Michaelis complex but also influence the rates of ensuing chemical steps. Only a small fraction of the available binding energy was exploited in interactions between substrate sidechains and the S1 and S1’ subsites, which indicates a modest level of complementarity. There was no correlation between S1 and S1’ specificities and amino acid abundance in hemoglobin. Interactions between PfA-M1 and the backbone atoms of the P1’ and P2’ residues as well as the P2’ sidechain further contributed to the catalytic efficiency of substrate hydrolysis. By demonstrating the engagement of multiple, broad-specificity subsites in PfA-M1, these studies provide insight into how this enzyme is able to efficiently generate amino acids from highly sequence-diverse di- and oligopeptides in the food vacuole.

A Naturally Variable Residue in the S1 Subsite of M1 Family Aminopeptidases Modulates Catalytic Properties and Promotes Functional Specialization

“…An "archetypal" S1 specificity can be loosely defined as a preference for the basic residues Arg and Lys and non-␤-branched nonpolar residues over acidic, small polar and ␤-branched nonpolar residues and proline. This specificity is observed for numerous well characterized enzymes, including mammalian aminopeptidase N, prokaryotic PepN, and PfA-M1 (13)(14)(15)(16). There are, however, many examples of deviations from this archetypal specificity.…”

“…Expression was induced with 1 mM isopropyl ␤-D-1-thiogalactoside for 4 h at 25°C. Cell pellets were lysed, and the proteins were purified and treated with tobacco etch virus protease as described previously for wild-type PfA-M1 (14). Purified enzymes were snap-frozen in liquid nitrogen and stored at Ϫ80°C.…”

“…Determination of Zn(II) stoichiometries for purified PfA-M1 and PepN variants was conducted by ion chromatography as described previously (14).…”

“…For each enzyme-substrate combination, assays to determine steady-state kinetic parameters were conducted using substrate concentrations ranging from ϳ0.2 to ϳ5 K m . Amino acid products were derivatized with AccQ⅐Tag (Waters) and analyzed by reverse phase ultrahigh pressure liquid chromatography as described previously (14). Peak areas for the AccQ⅐Tag derivative of Ala (all X-Ala substrates) or Leu (Gly-Leu substrate) were determined using Empower2 software (Waters) and were converted to picomoles of amino acid by reference to an Ala or Leu standard.…”

“…In most cases, data sets consisted of 9 substrate concentrations (including [S] ϭ 0) with a minority of data sets having 6 -8 substrate concentrations. Steady-state kinetic parameters for wild-type PfA-M1 were reported previously (14).…”