Analysis of the Mechanism of the Serratia Nuclease Using Site-Directed Mutagenesis

• DOI: 10.1093/nar/24.14.2632
• Publication Date: 2002-07-26T22:30:20Z
• Authors: P. Friedhoff; B. Kolmes; O. Gimadutdinow; W. Wende; K. L. Krause; A. Pingoud
• Keywords: Nuclease; Phosphodiester bond; Alanine; Site-directed mutagenesis; Enzyme Kinetics

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment six related nucleases, conserved amino acid residues that are located in proximity to previously identified catalytic site residue His89 were selected for mutagenesis study. Five out 12 analyzed turned be particular importance activity enzyme: Arg57, Arg87, His89, Asn119 Glu127. Their replacement by alanine, example, resulted mutant proteins very low activity, < 1% wild-type enzyme. Steady-state kinetic demonstrates some these mutants predominantly affected their kcat, others Km. These results determination pH metal ion dependence used tentative assignment function mechanism phosphodiester bond cleavage nuclease.