KINETIC-ANALYSIS OF THE CLEAVAGE OF NATURAL AND SYNTHETIC SUBSTRATES BY THE SERRATIA NUCLEASE

The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA a nd RNA with high specific activity. Steady-state and presteady-state k inetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Se rratia nuclease. Most of the natural substrates are cleaved with simil ar k(cat) and K-m values, the k(cat)/K-m ratios being comparable to th at of staphylococcal nuclease. Substrates with extreme structural feat ures. like poly(dA) . poly(dT) or poly(dG) . poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K-m, re spectively, as salmon testis DNA, Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inh ibition for the Serratia nuclease as reported recently. Experiments wi th short oligodeoxynucleotides confirmed previous results that for mod erately good cleavage activity the substrate should contain at least f ive phosphate residues. Shorter substrates are still cleaved by the Se rratia nuclease, albeit at a rate reduced by a factor of more than 100 . Cleavage experiments with oligodeoxynucleotides substituted by a sin gle phosphorothioate group showed that the negative charge of the pro- R(p)-oxygen of the phosphate group 3' adjacent to the scissile phospho diester bond is essential for cleavage, as only the R(p)-phosphorothio ate supports cleavage at the 5' adjacent phosphodiester bond. Furtherm ore, the modified bond itself is only cleaved in the R(p)-diastereomer , albeit 1000 times more slowly than the corresponding unmodified phos phodiester bond! which offers the possibility to determine the stereoc hemical outcome of cleavage. Pre-steady-state cleavage experiments dem onstrate that it is not dissocia tion of products but association of e nzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Ser ratia nuclease accepts thymidine 3',5'-bis(p-nitrophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and th ymidine 3'-(p-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smal ler than the rate of cleavage of macromolecular DNA or RNA.