Nucleic Acids Research, Vol 24, Issue 14 2632-2639, Copyright © 1996 by Oxford University Press
P Friedhoff, B Kolmes, O Gimadutdinow, W Wende, KL Krause and A Pingoud
Based on crystal structure analysis of the Serratia nuclease and a sequence
alignment of six related nucleases, conserved amino acid residues 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 importance for the
catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127.
Their replacement by alanine, for example, resulted in mutant proteins of
very low activity, < 1% of the activity of the wild-type enzyme.
Steady-state kinetic analysis of the mutant proteins demonstrates that some
of these mutants are predominantly affected in their kcat, others in their
Km. These results and the determination of the pH and metal ion dependence
of selected mutant proteins were used for a tentative assignment for the
function of these amino acid residues in the mechanism of phosphodiester
bond cleavage by the Serratia nuclease.
ARTICLES
Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis
Institut fur Biochemie, Justus-Liebig-Universitat, Giessen, Germany.
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