Inside the cells, proteins are surrounded by mixtures of different osmolytes. However, our current understanding of the combinatorial effects of such mixtures on the stability of proteins remains elusive. In the present study, the stability and structure of recombinant pyrazinamidase (PZase) from Mycobacterium tuberculosis were analyzed in the presence of stabilizing osmolytes (sorbitol, sucrose and glycerol) and alcohols (methanol, ethanol, isopropanol and n-propanol). The far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and thermostability results indicated that methanol, unexpectedly, has stronger effect on destabilization of the enzyme compared to ethanol which has larger log P. Interestingly, the relative half-life of PZase was longer in mixtures methanol with the osmolytes, sorbitol or sucrose (expectedly), or glycerol (unexpectedly), compared to other alcohols. Molecular dynamics simulation results showed that methanol increases the flexibility of region 5-40 and loop 51-71 in the PZase, which are potentially crucial for the stability and activity of the enzyme, respectively. Our results indicated that methanol can interact with PZase via hydrophobic interactions and hydrogen bonds, and therefore resulting in destabilization of the structure of the enzyme. In addition, glycerol probably increases the stability of the enzyme in methanol by disrupting the unfavorable hydrophobic interactions and hydrogen bonds. (C) 2017 Elsevier B.V. All rights reserved.
