Enhancement of enzyme activity through three-phase partitioning: crystal structure of a modified serine proteinase at 1.5 A resolution.

Three-phase partitioning is fast developing as a novel bioseparation strategy with a wide range of applications including enzyme stability and enhancement of its catalytic activity. Despite all this, the enzyme behaviour in this process still remains unknown. A serine proteinase, proteinase K, was subjected to three-phase partitioning (TPP). A 3 ml volume of proteinase K solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0) was brought to 30% (w/v) ammonium sulphate saturation by addition of saturated ammonium sulphate. tert-Butanol (6 ml) was added to this solution and the mixture was incubated at 25 degrees C for 1 h. The precipitated protein in the mid-layer was dissolved in 3 ml of 0.05 M acetate buffer, pH 6.0. The specific activity of the processed enzyme was estimated and was found to be 210% of the original enzyme activity. In order to understand the basis of this remarkable enhancement of the enzyme activity, the structure of the TPP-treated enzyme was determined by X-ray diffraction at 1.5 A resolution. The overall structure of the TPP-treated enzyme is similar to the original structure in an aqueous environment. The hydrogen bonding system of the catalytic triad is intact. However, the water structure in the substrate binding site has undergone a rearrangement as some of the water molecules are either displaced or completely absent. Two acetate ions were identified in the structure. One is located in the active site and seems to mimic the role of water in the enzyme activity and stability. The other is located at the surface of the molecule and is involved in stabilizing the local structure of the enzyme. The most striking observation in respect of the present structure pertains to a relatively higher overall temperature factor (B = 19.7 A(2)) than the value of 9.3 A(2) in the original enzyme. As a result of a higher B-factor, a number of residues, particularly their side chains, were found to adopt more than one conformation. It appears that the protein exists in an excited state which might be helping the enzyme to function more rapidly than the original enzyme in aqueous media. Summarily, the basis of increased enzymatic activity could be attributed to (i) the presence of an acetate ion at the active site and (ii) its excited state as reflected by an overall higher B-factor.