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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 K(m) and k(cat), 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.

SUBMITTER: Dalal S

PROVIDER: S-EPMC3307885 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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

Dalal Seema S Ragheb Daniel R T DR Klemba Michael M

Molecular and biochemical parasitology 20120213 1

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 an ...[more]

PMID: 22348949

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Project description:BackgroundThe Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.MethodsBy using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.ResultsThe high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.ConclusionsThe PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.

Dataset Information

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

Publications

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

Similar Datasets

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