Two Identical Subunits of the EcoRI Restriction Endonuclease Co‐operate in the Binding and Cleavage of the Palindromic Substrate

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Abstract
The cleavage of radioactively labeled double-stranded d(G-G-A-A-T-T-C-C) was studied in single turnover experiments with substrate and enzyme both being in the micromolar range. The reaction rate increased with enzyme concentration until a ratio of 1 tetrameric enzyme to 2 double-stranded substrates was reached, further increase of the enzyme concentration then leads to a sharp decline of the reaction rate. Apparently, 2 subunits of the EcoRI endonuclease cooperate in binding and possibly also in cleaving the palindromic substrate. The enzymatic action of the EcoRI endonuclease is inhibited by excess enzyme, possibly due to unspecific binding of the enzyme-substrate complex. The self-inhibition of EcoRI endonuclease was also observed with macromolecular substrates.