Relating Thermal/Colloidal Properties to Enzymatic Activity in the Intermediate and Native States of Amino Ester Hydrolase and Horseradish Peroxidase

While some proteins unfold according to the Lumry-Eyring model, many enzymes do not and instead exhibit more complex unfolding mechanisms. Enzymes that deviate from the Lumry-Eyring model may have intermediate states that exist between the native and unfolded state. Understanding the temperature dependence of the activity and stability of enzymes in their intermediate states may be crucial to optimizing their biotechnological and/or pharmaceutical applications. Horseradish peroxidase (HRP) and amino ester hydrolase (AEH) are enzymes observed to exhibit intermediate states in their unfolding. HRP has wide-ranging significance in biotechnology research for its utility as a reporter enzyme, in biocatalysis, and in bioremediation systems. AEH has garnered interest as a catalyst in the production of semi-synthetic beta-lactam antibiotics. A comprehensive characterization and comparison of either enzyme’s native state to its intermediate state has yet to be undertaken. Thermal transition temperatures for each enzyme were identified from melting curves measured by nano-differential scanning fluorimetry (nanoDSF). Dynamic light scattering (DLS) experiments were performed at temperatures corresponding to the native states and intermediate states of each enzyme to obtain particle size distributions and diffusion interaction parameters k_D. In addition, HRP activity was measured in the native and intermediate state by utilizing a colorimetric assay. AEH activity in each state was measured using ampicillin hydrolysis. Preliminary results suggest thermal transition temperatures from the native state to the intermediate state of approximately 33˚C and 42˚C for AEH and HRP respectively. Additionally, HRP activity has been found to significantly decrease in the intermediate state compared to the native state.