Malaria remains one of the most important infectious diseases, causing more than 200 million cases and 400,000 deaths each year. Plasmodium falciparum is the most lethal species and accounts for most of the malaria cases. Novel drugs are considered necessary owing to the resistance to the current treatments, artemisinin combination therapies (ACTs). Previous work in the Tilley lab confirmed that P. falciparum tyrosyl-tRNA synthetase (PfYRS) is the target of ML901, a nucleoside sulfamate derivative. ML901 acts via an unusual reaction hijacking mechanism that involves nucleophilic attack of the sulfamate nitrogen on the activated oxy-ester bond of the enzyme bound tyrosyl-tRNA to form an ML901-Tyr adduct. Importantly, human tyrosyl-tRNA (HsYRS) is not susceptible to nucleophilic attack by ML901, which enables highly specific targeting of plasmodium parasites. The structure of recombinant PfYRS was solved revealing a well-resolved 247KMSKS251 loop that encloses the active site. In comparison, the corresponding KMSSS loop of human tyrosyl-tRNA (HsYRS) is unresolved. Our hypothesis is that the flexibility of the loop is important for determining the potency of the inhibitor. Here, PfYRS was “humanized” by making a K250S mutant. Following optimization of the expression protocol, biophysical characterization revealed well-folded dimers. Thermal stabilization studies revealed apparently tighter binding of the AMP-Tyr intermediate. The consumption of ATP, i.e., the formation and release of AMP-Tyr in the initial reaction phase, was lower in PfYRS(K250S) than wildtype PfYRS, consistent with tighter binding of the AMP-Tyr intermediate. Generation of the ML901-Tyr adduct in the active site of wildtype PfYRS leads to marked thermal stabilization. By contrast, PfYRS(K250S) is less well stabilized indicating a reduced ability to generate the ML901-Tyr adduct. Analysis of the aminoacylation will confirm the molecular basis for the differential behavior of PfYRS(K250S) and wildtype PfYRS.