Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution

For the first time, it is demonstrated that exposure of an enzyme to anhydr ous organic solvents at optimized high temperature enhances its catalytic p ower through local changes at the binding region. Six enzymes, namely, prot einase K, wheat germ acid phosphatase, alpha -amylase, beta -glucosidase, c hymotrypsin and trypsin were exposed to acetonitrile at 70 degreesC for thr ee hr. The activities of these enzymes were found to be considerably enhanc ed. In order to understand the basis of this change in the activity of thes e enzymes, proteinase K was analyzed in detail using X-ray diffraction meth od. The overall structure of the enzyme was found to be similar to the nati ve structure in aqueous environment. The hydrogen bonding system of the cat alytic triad remained intact after the treatment. However, the water struct ure in the substrate binding site underwent some rearrangement as some of t he water molecules were either displaced or completely absent. The most str iking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were l ocated in the recognition site. Interlinked through water molecules, the si tes occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1 ) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the r ecognition sire introduced a striking conformation change in Ile 107 by rot ating its side chain about C alpha -C beta bond by 180 degrees to bring abo ut the delta -methyl soup within the range of attractive van der Waals inte ractions with the methyl group of CCN1. A similar change had earlier been o bserved in proteinase K when it was complexed to a substrate analogue, lact oferrin fragment.