Fluorescence quenching study of equilibrium binding of phage T4 Dam DNA-[N-6-adenine]-methyltransferase with substrates and ligands

Selective fluorescence quenching by iodide ions was used to evaluate the surface accessibility of tryptophan residues in T4 Dam DNA methyltransferase (T4 MTase) [EC 2.1.1.72]. Two of the three MTase tryptophans proved fully accessible for iodide ions, suggesting that they are located near the surface of the molecule. Quenching of intrinsic fluorescence of T4 MTase tryptophans was used to determine the parameters of enzyme binding with substrates (20-bp oligonucleotide duplex with a GATC site, and S-adenosyl-L-methyonine (SAM)) and substrate analogs (duplex without a GATC site and S-adenosyl-L-homocysteine (SAH)). Enzyme binding with small ligands (SAM and SAH) conforms with the modified Stern-Volmer equation applicable to 1:1 complexes. The dissociation constants for SAM and SAH were 0.24 and 2.3 mu M, respectively. In both cases, about one third of the total MTase fluorescence could be quenched by these ligands. Saturating binding of oligonucleotide duplexes resulted in 12% (oligonucleotide with GATC) or 19% (without GATC) quenching. In both cases, experimental quenching curves did not fit the modified Stern-Volmer equation, suggesting that the mechanism of oligonucleotide binding is more complex than that for small ligands.