Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96h, showed moderate tolerance in acetonitrile (45% activity after 72h) and much lower stability in methanol (40% activity after 24h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 degrees C as indicated by secondary structure monitoring, it was functionally stable only up to 45 degrees C, implicating thermolabile active site geometry. In GdnHCl [1.0M], 75% of the activity of KLKp was retained after incubation for 4h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.

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