ANALYSIS OF THE MECHANISM OF THE SERRATIA NUCLEASE USING SITE-DIRECTED MUTAGENESIS

Based on crystal structure analysis of the Serratia nuclease and a seq uence alignment of six related nucleases, consented amino acid residue s that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular import ance for the catalytic activity of the enzyme: Arg57, Arg87, His89, As n119 and Glu127. Their replacement by alanine, for example, resulted i n mutant proteins of very low activity, <1% of the activity of the wil d-type enzyme. Steady-state kinetic analysis of the mutant proteins de monstrates that some of these mutants are predominantly affected in th eir k(cat), others in their K-m. These results and the determination o f the pH and metal ion dependence of selected mutant proteins were use d for a tentative assignment for the function of these amino acid resi dues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.