(147f) Impact of Pegylation on Asparaginase Structure, Dynamics, and Thermodynamics from Molecular Dynamics Simulations

Asparaginase (ASNase) is widely used to treat diseases such as Acute Lymphoblastic Leukemia (ALL), but its clinical efficacy is often hindered by issues such as instability and immunogenicity. PEGylation – the covalent attachment of polyethylene glycol (PEG) – is an FDA-approved method commonly employed to overcome these limitations. Commercially available ASNase products are PEGylated via reaction with free amine groups on lysine residues, a non-specific approach that results in heterogeneous PEG attachment. This lack of specificity leads to PEGylated enzymes with similar molecular weights but varying conjugation sites, resulting in inconsistent enzymatic activity and therapeutic efficacy. Moreover, such heterogeneity complicates understanding how specific PEGylation sites affect enzyme dynamics and substrate binding mechanisms, knowledge critical for improving the consistency and effectiveness of PEGylated biologic therapeutics.

In this study, we apply all-atom molecular dynamics (MD) simulations to systematically investigate how mono-PEGylation (5 kDa) impacts ASNase dynamics and its binding affinity with L-Asparagine (Asn). By leveraging principal component analysis (PCA) and residue-level contact analysis, we characterize the alternation of essential dynamics caused by PEG attachment at different lysine residues in monomeric ASNase, emphasizing the influence of the PEGylation site. Furthermore, we employ funnel metadynamics (FM) to perform comprehensive binding energy analyses, revealing how PEG-induced dynamic alterations directly modulate substrate binding affinity and conformation of an enzyme. Our findings provide a thorough understanding of the complex interplay between PEGylation site, enzyme dynamics, and substrate binding affinity, offering valuable guidance for the rational design of more consistent and therapeutically optimized PEGylated enzymes.