Understanding the molecular basis of substrate specificity of an enzyme is very crucial to its functional importance in any biological system. Previously, we had experimentally validated a novel phenomenon using an enzyme engineering approach, to alter the substrate specificity via modification of the electrostatic properties of the gatekeeper residues in succinyl-CoA synthetase (SCS) of Blastocystis. The multiple sequence alignment (MSA) of SCSβ subunits from phylogenetically diverse organisms, depicted P. falciparum SCS to club with ADP-forming human, bovine and murine SCS enzymes. In the present study, we have explored the gatekeeper residues of P. falciparum SCS (PfSCS), an enzyme crucial for the generation of a metabolic intermediate- succinyl-CoA, required during the mosquito stages of the Plasmodium. With the construction of various gatekeeper mutants, structural modeling and enzyme kinetics experiments, our study concluded that PfSCS is an ADP-forming enzyme (KmATP=48 µM). Introduction of the exclusively charged- positive (Lys & Lys) & negative gatekeeper residues (Glu & Asp), demonstrated significant reductions in the ATP affinity, while no significant GDP-forming potential was recorded. Interestingly, simultaneous refolding of the nucleotide-binding site containing PfSCSβ subunit with the Blastocystis SCSα produced the active enzyme conformation. Therefore, the present study concluded that only electrostatic interactions at the gatekeeper region are not sufficient enough to alter the substrate specificity in PfSCS, as in case of Blastocystis SCS and further structural analysis is warranted with particular focus on the binding site architecture of PfSCS.