Analysis of the reaction mechanism of the non-specific endonuclease of Serratia 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 His89 is the general base and a Mg2+ ion bound to Glu127 the general acid in the mechanism of phosphodiester bond hydrolysis by the Serratia nuclease, and that Asn119 directly participates in catalysis, for example by transition state stabilisation. Arg57, Arg87 and Arg131, 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.
Kolmes
B
B
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Giessen, Germany.
Franke
I
I
Friedhoff
P
P
Pingoud
A
A
D004247:DNA / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D009596:Nitrophenols / Q000378:metabolism; D012706:Serratia marcescens / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism*
D009596:Nitrophenols; D013942:Thymine Nucleotides; C102666:deoxythymidine 3',5'-bis-(4-nitrophenylphosphate); D004247:DNA; D004492:Edetic Acid; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease; D008274:Magnesium; C024836:4-nitrophenol