(508g) The Role of Primary Sequence and Secondary Structure in the Hydrolysis of Peptide Bonds in Proteins

Proteins stored in liquid formulations are susceptible to a wide range of degradation pathways. Due to the requirement for high purity (>95%) throughout the shelf-life (1-2 years) of a pharmaceutical product, even these relatively slow reactions can drastically impact the feasibility of turning a drug candidate into a successful drug product. A significant source of fragmentation in proteins is the non-enzymatic hydrolysis of the peptide backbone. This reaction decreases the amount of active proteins in solution and produces new species which can enhance the rate of other degradation pathways such as aggregation. The primary sequence, the secondary structure and the pH have all been identified as important factors in determining the rate of hydrolysis. However, a molecular level understanding of the role that these factors play in the hydrolysis mechanism is still lacking. In the present study we employ high-level ab initio quantum chemistry calculations and machine learning algorithms to probe this complex mechanism

Experimental work indicates that a number of residues may promote hydrolysis. However, aspartic acid is typically the most susceptible and the combination of Asp–Pro is particularly sensitive. Therefore we have initially focused our attention on the mechanistic aspects of Asp–Pro hydrolysis. Through the use of ab initio quantum chemistry methods, we investigated the hydrolysis of this amino acid sequence in tripeptides systems. Our results show that the side chain of the aspartic acid interacts strongly with a neighboring water molecule during the rate-limiting step. This interaction promotes the disassociation of the water molecule, which then adds to the protein backbone. Our results concerning the role of proline in promoting hydrolysis show that it does not lower the enthalpy of the reaction. Our current hypothesis is that it lowers the activation energy for the hydrolysis reaction through inductive effects. Specifically, because proline is the only naturally occurring amino acid containing a secondary amine, it may promote the stabilization of the transition structure through charge delocalization.

These results shed light on the role of primary sequence, but because of the model systems are relatively small the effect of secondary and tertiary structure is not adequately accounted for. In order to investigate these, we gain insight by performing classical molecular dynamics simulation of whole proteins. Ensembles of protein conformations are then correlated to known aspects of the mechanism. Initial results from the simulation of an immunotoxin suggest that secondary structure elements affect rates of hydrolysis through two primary means – steric interactions and hydrogen bonding.