title: Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease.
abstract: We have used the intrinsic tryptophan fluorescence of the EcoRV restriction endonuclease to monitor changes in protein conformation during binding and cleavage of a duplex oligodeoxynucleotide substrate. Appropriate conditions for single-turnover reactions were first determined by steady-state kinetics. When single turnovers were monitored by stopped-flow fluorescence, the mixing together of EcoRV, oligonucleotide and MgCl2 resulted in a rapid increase in tryptophan fluorescence followed by a slow decrease. Further analysis by order-of-mixing and quench experiments showed that the transient increase in fluorescence was due to a conformational change coupled to DNA binding, while the subsequent decay was concomitant with phosphodiester hydrolysis. The rate of the latter step varied with the concentration of Mg2+ ions, but another Mg(2+)-dependent transition was observed upon the addition of MgCl2 to a preformed enzyme-DNA complex. These results lead to a reaction scheme in which one Mg2+ binds to the active site prior to phosphodiester hydrolysis but a second Mg2+ is then needed to carry out the hydrolytic reaction. This scheme is correlated to the crystal structures of the EcoRV endonuclease and its complexes with DNA and Mg2+ ions.
authors: Department of Biochemistry, University of Bristol, U.K.
authors: G S
authors: GS
authors: Baldwin
authors: I B
authors: IB
authors: Vipond
authors: S E
authors: SE
authors: Halford
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D014364:Tryptophan / Q000378:metabolism
chemical_list: D014364:Tryptophan; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases