On the Role of Protein Structural Dynamics in the Catalytic Activity and Thermostability of Serine Protease Subtilisin Carlsberg

摘要

The effect of modulating the structural dynamics by incremental glycosylation on enzyme activity and thermostability has thus far only been investigated in detail for the serine protease α-chymotrypsin (for a recent review see Solá et al., Cell. Mol. Life Sci. 2007, 64(16):2133–2152). Herein, we extend this type of study to a structurally unrelated serine protease, specifically, subtilisin Carlsberg. The protease was glycosylated with activated lactose and various lactose-subtilisin conjugates were obtained and characterized. Near UV-CD spectroscopy revealed that the tertiary structure was unaffected by the procedure. H/D exchange FT-IR spectroscopy was performed to assess the structural dynamics of the enzyme. It was found that increasing the level of glycosylation caused a linearly dependent reduction in structural dynamics. This led to an increase in thermostability and a decrease in the catalytic turnover rate for both, acylation and de-acylation step. These data highlight the possibility that a structural dynamics – activity relationship might be a phenomenon generally found in serine proteases.