Profiling Enzyme Activity of l-Asparaginase II by NMR-Based Methyl Fingerprinting at Natural Abundance

Abstract

L-asparaginase II (MW 135 kDa) from E. coli is an FDAapproved 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, methylbased 2D [ 1 H-13 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 [ 1 H-13 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.

Exploring the Higher Order Structure and Conformational Transitions in Insulin Microcrystalline Biopharmaceuticals by Proton-Detected Solid-State Nuclear Magnetic Resonance at Natural Abundance

“…The latest guidelines for developing biosimilars strongly recommend reporting on the folding and aggregation properties, as well as characterizing higher order structures (HOSs). Methyl groups, in particular, have been extensively used in biomolecular NMR as highly sensitive site-specific probes for structure, − dynamics, , and interaction studies. − Due to their well-dispersed nature in the protein’s 3D structure, methyl chemical shifts can provide valuable information on subtle alterations inside the hydrophobic pocket of the protein that can alter the native folding as well as potential changes in the oligomerization state of a protein. Alternatively, these can also be explored for identifying binding epitopes in the protein–receptor interactions .…”