Kinetics of the uracil-DNA glycosylase/inhibitor protein association. Ung interaction with Ugi, nucleic acids, and uracil compounds.
The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) inactivates Escherichia coli uracil-DNA glycosylase (Ung) by forming an Ung.Ugi protein complex with 1:1 stoichiometry. Stability of the Ung.Ugi complex was demonstrated by the inability of free Ugi to exchange with Ugi bound in preformed complex. Ung was reacted with fluorescein 5-isothiocyanate to produce fluorescent-Ung (F-Ung), which retained full uracil-DNA glycosylase activity and susceptibility to Ugi inactivation. Addition of Ugi to F-Ung under steady-state conditions resulted in saturable (15%) fluorescence quenching at a F-Ung.Ugi ratio of 1:1.4. Dissociation constants determined for the F-Ung interaction with M13 DNA, uracil-containing DNA, and poly(U) equaled 600, 220, and 190 microM, respectively. While F-Ung associated with nucleic acid polymers was able to bind Ugi efficiently, F-Ung bound in the F-Ung.Ugi complex could no longer effectively bind nucleic acid. Stopped-flow kinetic analysis suggested the F-Ung/Ugi association was described by a two-step mechanism. The first step entailed a rapid pre-equilibrium distinguished by the dissociation constant Kd = 1.3 microM. The second step led irreversibly to the formation of the final complex and was characterized by the rate constant k = 195 s-1. We infer Ugi inactivates Ung through the formation of an exceptionally stable protein-protein complex.
Bennett
S E
SE
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
Schimerlik
M I
MI
Mosbaugh
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DW
D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D016650:Fluorescein-5-isothiocyanate; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics; D009696:Nucleic Acids / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase; D014764:Viral Proteins / Q000378:metabolism*
D009696:Nucleic Acids; D014764:Viral Proteins; C027619:uracil-DNA glycosylase inhibitor protein, B. subtilis bacteriophage; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D016650:Fluorescein-5-isothiocyanate