l-asparaginase
II (MW 135 kDa) fromE.
coliis 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 [1H-13C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural
abundance, has been used to profile the enzymatic activity of the
commercially available enzyme drug. The [1H-13C]-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.