Asparaginase (ASNase) is widely utilized in the treatment of diseases such as Acute Lymphoblastic Leukemia (ALL). However, its clinical effectiveness is often compromised by instability and immunogenicity. PEGylation – the covalent attachment of polyethylene glycol (PEG) – is an FDA-approved strategy frequently employed to address these limitations. While several PEGylated ASNase formulations are commercially available, existing PEGylation methods typically target free amine groups on lysine residues. This approach generates heterogeneous conjugation sites, leading PEG to attach at multiple lysine residues without site specificity, especially when multiple reactive lysine residues are available. This non-specific approach results in heterogeneous populations of PEGylated enzymes with identical molecular weights but variable attachment sites, potentially leading to differences in enzymatic activity and therapeutic efficacy. Such heterogeneity complicates a fundamental understanding of how specific PEGylation sites influence essential enzyme dynamics and substrate binding mechanisms. This knowledge is crucial for enhancing the consistency and efficacy of PEGylated biologic therapeutics.
In this study, we employ all-atom molecular dynamics (MD) simulations to systematically evaluate the impact of mono PEGylation (5kDa) on ASNase dynamics and substrate binding interaction with L-Asparagine (Asn). Principal component analysis (PCA) and residue-level contact analysis allow characterization of essential dynamic shifts across different PEGylation sites on monomeric ASNase, highlighting the significant influence of PEG attachment location. Furthermore, comprehensive binding energy analyses, conducted using funnel metadynamics (FM), demonstrates how these PEG-induced dynamic changes directly influence substrate binding affinity and alter the binding conformation. Our findings offer an in-depth understanding of the intricate relationship between PEGylation site specificity, enzyme essential dynamics, and binding affinity, thereby guiding rational design strategies toward more consistent and therapeutically optimized PEGylated enzymes.
