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 intermedius, we used site-directed mutagenesis, On cleavage of various substrates 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 in activity is determined preferentially by the decrease in the molecular rate constant k(cat) with a relatively small change of enzyme-substrate affinity, characterized by K-m, This is the expected result if Arg(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 have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg(82) by alanine is mediated by the effect of Arg(82) on the pK of catalytic residues. (C) 1998 Federation of European Biochemical Societies.