KINETIC MODELING OF THE MECHANISM OF ALLOSTERIC INTERACTION OF RESTRICTION-ENDONUCLEASE ECORII WITH 2 DNA SITES

Allosteric activation of the restriction endonuclease EcoRII was studi ed experimentally and theoretically. Cleavage of one substrate in the presence of another substrate was monitored. The dependence of the all osteric activation of cleavage during mutual substrate induction on th e ratio of kinetic parameters of the enzyme interaction with each subs trate was determined. Pairs of DNA duplexes with different kinetic con stants for the studied enzyme were constructed and were used as substr ates or substrate analogs. A kinetic mechanism is suggested that chara cterizes allosteric influence of two DNA recognition sites on the dime ric EcoRII molecule. Mathematical modeling was used to analyze the kin etic mechanism and to evaluate optimal characteristics of the inducer substrate. EcoRII was activated when: 1) the substrate concentration i s decreased; 2) the binding capacity of two substrate or two inducer m olecules with the enzyme is decreased; 3) the enzyme binding of one su bstrate molecule is enhanced versus binding of one inducer molecule; 4 ) the k(cat) of the enzyme-substrate complex, which comprises a substr ate molecule and an inducer molecule, is increased.