CONTRIBUTION OF ARGININE-82 AND ARGININE-86 TO CATALYSIS OF RNASES FROM BACILLUS-INTERMEDIUS (BINASE)

To elucidate the functional role of Arg(82) and Arg(86) in the enzyme activity of binase, the extracellular ribonuclease of Bacillus interme dius, we used site-directed mutagenesis, On cleavage of various substr ates the catalytic activity of binase mutant Arg(86)Ala is 2.7 x 10(3) -7.7 x 10(3) times less than that of the native enzyme. The decrease i n activity is determined preferentially by the decrease in the molecul ar rate constant k(cat) with a relatively small change of enzyme-subst rate affinity, characterized by K-m, This is the expected result if Ar g(86) acts to lower the energy of a transition state of the reaction. The replacement of Arg(82) by Ala causes a 5-19-fold activity decrease , depending on the substrate, We propose that this residue does not ha ve a direct catalytic function in the molecular mechanism of the binas e action and that the activity decrease of binase on the replacement o f Arg(82) by alanine is mediated by the effect of Arg(82) on the pK of catalytic residues. (C) 1998 Federation of European Biochemical Socie ties.