Analysis of the reaction mechanism of the non‐specific endonuclease of Serratia marcescens using an artificial minimal substrate

[摘要]

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.

[学科分类] 生物化学/生物物理

[关键词] DNase 1;Staphylococcal nuclease;Phosphodiesterase;Enzyme mechanism;Acid base catalysis;Deoxythymidine 3′;5′-bis-(p-nitrophenyl- phosphate)