PROBING THE ROLE OF CYSTEINE RESIDUES IN THE ECOP15I DNA METHYLTRANSFERASE

Chemical modification using thiol-directed agents and site-directed mu tagenesis has been used to investigate the role of cysteine residues o f EcoP15I DNA methyltransferase, Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethy lmaleimide and iodoacetamide, 5,5'-Dithiobis(2-nitrobenzoic acid) titr ation of the enzyme under nondenaturing and denaturing conditions conf irmed the presence of six cysteine residues without any disulfides in the protein. An are that relatively bulky reagents inactivate the meth yltransferase by directly occluding the substrate-binding site or by l ocking the methyltransferase in an inactive conformation, we used site -directed mutagenesis to sequentially replace each of the six cysteine s in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes re tained significant activity as assessed by in vivo and in vitro assays . The effects of the substitutions on the function of EcoP15I DNA meth yltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis, Our resu lts clearly indicate that the cysteines at positions other than 344 ar e not essential for activity. In contrast, the C344A enzyme showed a m arked loss of enzymatic activity. More importantly, whereas the inacti ve C344A mutant enzyme bound S-adenosyl-L-Methionine, it failed to bin d to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken togethe r, these results convincingly demonstrate that the Cys-344 is necessar y for enzyme activity and indicate an essential role for it in DNA bin ding.