Kinetic Studies on the Irreversible Inhibition of Restriction Endonuclease Pst I by Site-Specific Inhibitors

The irreversible modifying effects on Pst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou,C.L. Pyridoxal phosphate (PLP), p chloromercuribenzoic acid (PCMB),diisopropyl fluorophosphate (DFP), 2,3 diacetyl (DAC) and N ethyl 5 phenylisoxazoliun 3’ sulfonate (woodward’s reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity of Pst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant, A and the microcosmic inhibition rate constants, k +0 and k′ +0 of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn’t the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration of Pst I conformation and then influence the ability of Pst I recognizing and incising DNA specifically.