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Analysis of the reaction mechanism of the non‐specific endonuclease of Serratia marcescens using an artificial minimal substrate
Author(s) -
Kolmes Bettina,
Franke Ingo,
Friedhoff Peter,
Pingoud Alfred
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(96)01210-0
Subject(s) - serratia marcescens , substrate (aquarium) , substrate specificity , endonuclease , chemistry , biophysics , biology , biochemistry , escherichia coli , enzyme , ecology , gene
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 base and a Mg 2+ ion bound to Glu 127 the general acid in the mechanism of phosphodiester bond hydrolysis by the Serratia nuclease, and that Asn 119 directly participates in catalysis, for example by transition state stabilisation. Arg 57 , Arg 87 and Arg 131 , essential for nuclease activity, are not needed for cleavage of the artificial substrate, suggesting that they are involved in binding and positioning of nucleic acid substrates.