The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone
Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl‐CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been s...
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doaj-f5292120853846b49db0b3417ba198cd2021-03-04T10:35:45ZengWileyFEBS Open Bio2211-54632021-03-0111357858710.1002/2211-5463.13034The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues aloneKapil Vashisht0Pallavi Singh1Sonia Verma2Rajnikant Dixit3Neelima Mishra4Kailash C. Pandey5Protein Biochemistry and Engineering Lab, Parasite‐Host Biology Group ICMR–National Institute of Malaria Research New Delhi IndiaProtein Biochemistry and Engineering Lab, Parasite‐Host Biology Group ICMR–National Institute of Malaria Research New Delhi IndiaProtein Biochemistry and Engineering Lab, Parasite‐Host Biology Group ICMR–National Institute of Malaria Research New Delhi IndiaProtein Biochemistry and Engineering Lab, Parasite‐Host Biology Group ICMR–National Institute of Malaria Research New Delhi IndiaProtein Biochemistry and Engineering Lab, Parasite‐Host Biology Group ICMR–National Institute of Malaria Research New Delhi IndiaProtein Biochemistry and Engineering Lab, Parasite‐Host Biology Group ICMR–National Institute of Malaria Research New Delhi IndiaSubstrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl‐CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild‐type), generated by refolding of the individual P. falciparum SCSβ and Blastocystis SCSα subunits, demonstrated ADP‐forming activity (KmATP = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSβ subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required.https://doi.org/10.1002/2211-5463.13034gatekeeper residuesmalariaPlasmodium falciparumsite‐directed mutagenesissubstrate specificitysuccinyl‐CoA synthetase |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kapil Vashisht Pallavi Singh Sonia Verma Rajnikant Dixit Neelima Mishra Kailash C. Pandey |
spellingShingle |
Kapil Vashisht Pallavi Singh Sonia Verma Rajnikant Dixit Neelima Mishra Kailash C. Pandey The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone FEBS Open Bio gatekeeper residues malaria Plasmodium falciparum site‐directed mutagenesis substrate specificity succinyl‐CoA synthetase |
author_facet |
Kapil Vashisht Pallavi Singh Sonia Verma Rajnikant Dixit Neelima Mishra Kailash C. Pandey |
author_sort |
Kapil Vashisht |
title |
The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone |
title_short |
The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone |
title_full |
The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone |
title_fullStr |
The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone |
title_full_unstemmed |
The nucleotide specificity of succinyl‐CoA synthetase of Plasmodium falciparum is not determined by charged gatekeeper residues alone |
title_sort |
nucleotide specificity of succinyl‐coa synthetase of plasmodium falciparum is not determined by charged gatekeeper residues alone |
publisher |
Wiley |
series |
FEBS Open Bio |
issn |
2211-5463 |
publishDate |
2021-03-01 |
description |
Substrate specificity of an enzyme is an important characteristic of its mechanism of action. Investigation of the nucleotide specificity of Plasmodium falciparum succinyl‐CoA synthetase (SCS; PfSCS) would provide crucial insights of its substrate recognition. Charged gatekeeper residues have been shown to alter the substrate specificity via electrostatic interactions with approaching substrates. The enzyme kinetics of recombinant PfSCS (wild‐type), generated by refolding of the individual P. falciparum SCSβ and Blastocystis SCSα subunits, demonstrated ADP‐forming activity (KmATP = 48 µm). Further, the introduction of charged gatekeeper residues, either positive (Lys and Lys) or negative (Glu and Asp), resulted in significant reductions in the ATP affinity of PfSCS. It is interesting to note that the recombinant PfSCSβ subunit can be refolded to a functional enzyme conformation using Blastocystis SCSα, indicating the possibility of subunits swapping among different organisms. These results concluded that electrostatic interactions at the gatekeeper region alone are insufficient to alter the substrate specificity of PfSCS, and further structural analysis with a particular focus on binding site architecture is required. |
topic |
gatekeeper residues malaria Plasmodium falciparum site‐directed mutagenesis substrate specificity succinyl‐CoA synthetase |
url |
https://doi.org/10.1002/2211-5463.13034 |
work_keys_str_mv |
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