The p.V27A Variant of L-Asparaginase II Minimizes Glutaminase Activity Without Compromising Anti-Leukemic Function

L-Asparaginase is a fundamental chemotherapeutic agent for acute lymphoblastic leukemia (ALL) that depletes circulating asparagine to induce apoptosis in leukemic blasts. However, the clinical administration of Escherichia coli L-asparaginase is frequently limited by severe hepatotoxicity and neurotoxicity, which are directly driven by its off-target L-glutaminase co-activity. We hypothesized that rationally engineering the active-site flexible loop (ASFL), the dynamic motif that regulates substrate access, could decouple these catalytic activities. In this study, we present the structural and functional characterization of a novel E. coli L-asparaginase variant harboring a targeted V27A mutation within the ASFL. Biophysical investigations using CD, FTIR, Raman spectroscopy, and dynamic light scattering reveal that this substitution triggers a profound conformational shift, characterized by increased α-helical content and significant structural compaction. This structural tightening not only enhances the enzyme's thermodynamic resistance to chemical denaturation but also improves substrate selectivity. Remarkably, p.V27A demonstrates a nearly two-fold increase in the K_m for glutamine, significantly suppressing toxic off-target activity, while simultaneously lowering the K_m for asparagine to improve therapeutic catalytic efficiency. Crucially, in vitro cytotoxicity assays against Jurkat T-ALL cells confirm that p.V27A retains potent anti-leukemic efficacy comparable to the wild-type enzyme. Together, our findings establish that targeted modification of the ASFL through the clinically relevant V27A substitution alters substrate discrimination, particularly by reducing productive accommodation of the larger L-glutamine substrate, while preserving L-asparagine catalytic function. By mechanically tuning these loop dynamics to favor specificity, we demonstrate that the p.V27A variant delivers an improved safety profile without compromising therapeutic potency, emerging as a highly promising candidate for next-generation leukemia therapy.