l-asparaginase II (MW 135 kDa) from is an FDA-approved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, the structural basis of enzyme action, in solution, remains widely contested. In this work, methyl-based 2D [H-C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been used to profile the enzymatic activity of the commercially available enzyme drug. The [H-C]-HSQC NMR spectra of the protein reveal the role of a flexible loop segment in the activity of the enzyme, in solution. Addition of asparagine to the protein results in distinct conformational changes of the loop that could be signatures of intermediates formed in the catalytic reaction. To this end, an isothermal titration calorimetry (ITC)-based assay has been developed to measure the enzymatic reaction enthalpy, as a marker for its activity. Combining both ITC and NMR, it was shown that the disruption of the protein conformation can result in the loss of function. The scope, robustness, and validity of the loop fingerprints in relation to enzyme activity have been tested under different solution conditions. Overall, our results indicate that 2D NMR can be used reliably to gauge the structure-function of this enzyme, bypassing the need to label the protein. Such natural abundant NMR methods can be potentially extended to probe the structure-function aspects of high-molecular-weight protein therapeutics (glycosylated protein drugs, enzymes, therapeutic monoclonal antibodies, antibody-drug conjugates, and Fc-fusion proteins), where (a) flexible loops are required for their function and (b) isotope labeling may not be straightforward.