COMPARATIVE-STUDY OF BINASE AND BARNASE - EXPERIENCE IN CHIMERIC RIBONUCLEASES

Chimeric enzymes were constructed to elucidate the differences in phys icochemical properties of two related bacterial RNases, barnase and bi nase, Chimeras (Ba26Bi, Ba73Bi, Ba26Bi73Ba and Bi73Ba) contain six to thirteen residue substitutions relative to barnase, which are beyond t he active site. The catalytic activity of RNases toward GpU, GPC and p oly(I), as well as conformational distinctions and heat denaturation p arameters, were studied. Thermal denaturation of binase, barnase and c himeric RNases is a two-state transition. The mutation-induced changes in the free energy of unfolding of barnase deduced from thermal and u rea denaturation nearly coincide. The kinetic parameters for GpU and G pC demonstrate that the chimeras fall into two groups: barnase-like an d binase-like. This division is determined by the origin of their C-te rminal part (residues 73-110) which is also responsible for their ther mostability at pH 2.4. An inverse linear dependence was found between k(cat) for poly(I) and denaturation temperature of RNases at pH 5.5, w hich points out that certain lability of the protein molecule appears to be necessary for efficient polynucleotide cleavage.