MUTATIONAL ANALYSIS OF THE ACTIVE-SITE OF RNASE OF BACILLUS-INTERMEDIUS (BINASE)

To elucidate the functional role of some residues in the active site,o f binase, the extracellular ribonuclease of Bacillus intermedius, we u sed site-directed mutagenesis. On cleavage of various substrates the c atalytic activity of binase mutant His(101)Glu is 2.0-2.7% of that for native enzyme. The decrease in activity is determined mainly by the d ecrease in molecular rate constant k(cat), with almost unchanged affin ity of the enzyme for the substrate, characterized by K-M. This is the expected result if His(101) acts as an general acid, donating a proto n to the leaving group on cleavage of a phosphodiester bond. The repla cement of Lys(26) by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both k(cat) and K-M for poly(I) and poly(A) but in k(cat) a lone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.