Elucidating the chemical and thermal unfolding profiles of organophosphorus hydrolase and increasing its operational stability
Organophosphorus hydrolase (OPH, EC 18.104.22.168) is a homodimeric enzyme that has been observed to unfold via a three-state unfolding pathway (N2* ? I2 ? 2U) under chemical denaturing conditions. The dimeric intermediate (I2) is catalytically inactive and, although this enzyme has a very large overall conformational stability (~40 kcal/mol), it takes only a small amount of energy (~4 kcal/mol) to unfold this enzyme into its inactive form. So that this enzyme might be engineered as a more effective tool for nerve agent countermeasures and bioremediation purposes, its operational stability (the energy required to unfold the enzyme from its active, dimeric state to its inactive, dimeric state) must be increased. For this purpose, it is necessary to understand how the enzyme unfolds into its inactive, intermediate state. As tryptophan residues are sensitive probes of the microenvironment surrounding the residue, enzyme variants consisting of one tryptophan per subunit were constructed. Unfortunately, these variant enzymes did not fold into active conformations, and so could not be used to develop an accurate unfolding profile for the wild type enzyme. Limited proteolysis of OPH by thermolysin revealed detailed information on the unfolding process of OPH in chemical and thermal denaturing conditions. Mild denaturing conditions induced an initial enhancement of activity with a subsequent loss of catalytic activity upon more aggressive treatment. Under thermal conditions from 35 ? 55 ?C, the enzyme developed a well populated and active intermediate that displayed maximal activity. Similarly, the enzyme displayed maximal activity when incubated at 1.0 M urea. The regions of the enzyme, which became accessible to proteolysis at 45 ?C and 1 M urea, were identical. This suggested that increased flexibility of these regions was coupled with the increase in the enzyme?s catalytic activity. Two regions that were determined by limited proteolysis to be the first to unfold were bridged with a novel disulfide bond. The result was an enzyme with an increased operational stability and resistance to proteolysis. This enzyme retained approximately 70% of its original activity in 8 M urea while no activity remained for the wild type enzyme when incubated in 6.5 M urea.