Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease.
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.
Baldwin
G S
GS
Department of Biochemistry, University of Bristol, U.K.
Vipond
I B
IB
Halford
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SE
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
D014364:Tryptophan; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases