INCREASE OF SPECIFICITY OF RNASE FROM BACILLUS-AMYLOLIQUEFACIENS (BARNASE) BY SUBSTITUTION OF GLU FOR SER57 USING SITE-DIRECTED MUTAGENESIS

Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus i ntermedius show the specificity towards the nature of a nucleoside at the 03' end of the phoshodiester bond to be split in the preference or der G > A much greater than U > C in the cleavage reactions of polynuc leotides. It follows from the X-ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high-specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional h ydrogen bond between the protein and a purine base in the case of bact erial guanyl-preferring RNases in contrast to the high-specificity gua nylic RNases. To examine this hypothesis we prepared the Ser57 --> 4Gl u mutant of B. amyloliquefaciens, in which this hydrogen bond is elimi nated. Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35-times greater than that of the wild -type RNases from B. amyloliquefaciens and close to that of RNases T1.