Use of isotope-dilution phenomenon to advantage in the determination of kinetic constants Km and Kcat for BamHI restriction endonuclease: an empirical and iterative approach.

An assay using a very small amount of 35S-labeled deoxyoligonucleotide as a substrate for the determination of Km and Kcat for the restriction enzyme BamHI is described. Two synthetic deoxyoligonucleotides, ATGGCGGATCCGC and ATGGCGGAGCCGC, containing the cognate and a mismatch BamHI sequence, respectively, were labeled by an end-filling reaction using the Klenow fragment of DNA polymerase and [35S]dATP to generate the labeled self-complementary substrates. The dependence of BamHI hydrolysis on substrate concentration was investigated using mixtures of a fixed amount of radiolabeled substrate and varying amounts of cold-labeled substrate over a wide range. The apparent competitive inhibition observed due to the phenomenon of carrier dilution was analytically corrected by an empirical as well as an iterative approach to give Km values comparable to those reported in the literature. We have found that the values obtained using the empirical formula are very close to the precise values obtained through iteration. Our procedure has used isotopic dilution to advantage to make the assay less expensive and can be applied effectively to any enzyme-substrate reaction in which the substrate and the product have radioactive labels. The method would be especially useful for a rapid analysis and comparison of kinetic constants of various mutant enzymes or substrates.