Does Microsecond Active-Site Dynamics Primarily control Proteolytic Activity of Bromelain? Clues from Single Molecular Level Study with a Denaturant, a Stabilizer and a Macromolecular Crowder

Abstract

Proteins are dynamic entity with various molecular motions at different timescale and length scale. Molecular motions are crucial for the optimal function of an enzyme. It seems intuitive that these motions are crucial for optimal enzyme activity. However, it is not easy to directly correlate an enzyme's dynamics and activity due to biosystems' enormous complexity. amongst many factors, structure and dynamics are two prime aspects that combinedly control the activity. Therefore, having a direct correlation between protein dynamics and activity is not straightforward. Herein, we observed and correlated the structural, functional, and dynamical responses of an industrially crucial proteolytic enzyme, bromelain with three versatile classes of chemicals: GnHCl (protein denaturant), sucrose (protein stabilizer), and Ficoll-70 (macromolecular crowder). The only free cysteine (Cys-25 at the active-site) of bromelain has been tagged with a cysteine-specific dye to unveil the structural and dynamical changes through various spectroscopic studies both at bulk and at the single molecular level. Proteolytic activity is carried out using casein as the substrate. GnHCl and sucrose shows remarkable structure-dynamics-activity relationships. Interestingly, with Ficoll-70, structure and activity are not correlated. However, microsecond dynamics and activity are beautifully correlated in this case also. Overall, our result demonstrates that bromelain dynamics in the microsecond timescale around the active-site is probably a key factor in controlling its proteolytic activity.