초나선형 DNA(Form I)에 대한 Eco RI 제한 효소의 절단 반응은 한가닥만 잘린 open-circular 형태(Form II)의 중간 생성물을 거쳐 직선형 DNA(Form III)를 생성하며, 이 중간 생성물의 축적량은 최적 조건에서 벗어날수록 증가되었다. 이 효소의 Form I, II 그리고 III에 대한 절단 반응은 EtBr의 농도를 높이고, 온도를 낮출수록 저해되었으며, Form II와 Form III의 절단 반응이 Form I에 비하여 EtBr에 의한 저해가 심한 것으로 나타났다. 이 반응들은 낮은 농도의 NaCl, spermine 그리고 spermidine에 의하여 촉진되었고, 높은 농도에서는 현저히 감소되었다. 초나선형과 직선형 DNA의 Km값은 각각 5.8, 8.1nM을 보여 초나선형이 직선형보다 효소에 대한 친화력이 더 크다는 결과를 나타내었다. 효소의 활성은 diisopropylfluoro-phosphate, iodoacetamide 그리고 diethylpyrocarbonate에 민감한 저해를 보였고, trinitrobenzenesulfonate에 의해 저해 현상을 보임으로 효소의 lysine 잔기의 양전하가 DNA의 음전하와 상호작용하고, serine과 histidine 잔기가 촉매작용에 직접 관여한다는 사실을 제시할 수 있었다.
The closed circular form DNA (Form I) was changed into linear form DNA (form III) by Eco RI endonuclease through the intermediate open-circular form (form II), which could be introduced to the second turn-over, and the intermediate form was more accumulated when the condition was more deviated from the optimal state. The cleavage of form II and III DNA was more sensitive to inhibition to ethidium bromide (EtBr) than the linearization of form I DNA. The cleavage reactions were stimulated by a low concentration of NaCl, spermidine, and spermine. At high concentration, reactions were markedly inhibited by masking the charge of DNA. Km value of form I DNA was lower than that of form III DNA and thus superhelical form had a higher affinity than linear form DNA. The enzyme activity was sensitive to treatment of functional group blocking agent such as diisopropyl fluorophosphate, iodoacetamide, and diethylpyrocarbonate. The enzyme activity was also decreased in the presence of trinitrobenzene-sulfonate. From these results, it was thought that serine and histidine residues of the enzyme were directly involved in the phosphodiesterase action and the lysine residues might hold the phosphodiester bond of DNA.
