Impact of oxidation on protein therapeutics: conformational dynamics of intact and oxidized acid-β-glucocerebrosidase at near-physiological pH.

The solution dynamics of an enzyme acid-beta-glucocerebrosidase (GCase) probed at a physiologically relevant (lysosomal) pH by hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals very uneven distribution of backbone amide protection across the polypeptide chain Highly mobile segments are observed even within the catalytic cavity alongside highly protective segments, highlighting the importance of the balance between conformational stability and flexibility for enzymatic activity Forced oxidation of GCase that resulted in a 40-60% reduction in in vitro biological activity affects the stability of some key structural elements within the catalytic site These changes in dynamics occur on a longer time scale that is irrelevant for catalysis, effectively ruling out loss of structure in the catalytic site as a major factor contributing to the reduction of the catalytic activity Oxidation also leads to noticeable destabilization of conformation in remote protein segments on a much larger scale, which is likely to increase the aggregation propensity of GCase and affect its bioavailability Therefore, it appears that oxidation exerts its negative impact on the biological activity of GCase indirectly, primarily through accelerated aggregation and impaired trafficking