ANALYSIS OF THE REACTION-MECHANISM OF THE NONSPECIFIC ENDONUCLEASE OFSERRATIA-MARCESCENS USING AN ARTIFICIAL MINIMAL SUBSTRATE

We have studied the mechanism of action of the Serratia nuclease using deoxythymidine 3',5'-bis-(p-nitrophenyl-phosphate) as a substrate, A comparison of the activity with which the wild-type enzyme and several mutant enzymes attack this artificial substrate and herring sperm DNA , respectively, supports the suggestion that His(89) is the general ba se and a Mg2+ ion bound to Glu(127) the general acid in the mechanism of phosphodiester bond hydrolysis by the Serratia nuclease, and that A sn(119) directly participates in catalysis, for example by transition state stabilisation, Arg(57), Arg(87) and Arg(131), essential for nucl ease activity, are not needed for cleavage of the artificial substrate , suggesting that they are involved in binding and positioning of nucl eic acid substrates.