Kinetic Mechanism of Adenine Nucleotide Binding to and Hydrolysis by the Escherichia coli Rep Monomer. 2. Application of a Kinetic Competition Approach

Keith J.M. Moore, Timothy M. Lohman

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The Escherichia coli Rep protein is a DNA helicase that functions as a homodimer to catalyze the unwinding of duplex DNA during DNA replication in a reaction that is coupled to the binding and hydrolysis of ATP. As a first step toward a molecular understanding of the interactions of Rep with adenine nucleotides, we have investigated the kinetic mechanism of adenine nucleotide binding to the Rep monomer, which is the state of the protein in the absence of DNA. Although ATP binding to Rep does not significantly change the intrinsic tryptophan fluorescence, the binding of the fluorescent nucleotide analogue, 2′(3′)-O-(N-methylanthraniloyl)-ATP (mantATP) is associated with a large increase in mant nucleotide fluorescence intensity [λex = 290 nm, λem> 420 nm; Moore, K. J. M., & Lohman, T. M.(1994) Biochemistry (preceding article in this issue)]. We have used the fluorescence signal from mantATP binding to monitor the kinetics of nonfluorescent nucleotide binding to Rep by a kinetic competition approach. The simultaneous and parallel binding of a mixture of mantATP and ATP to the Rep monomer is associated with a complex triphasic fluorescence transient during the approach to equilibrium. Global analysis of the fluorescence transients over a range of [ATP] by numerical integration techniques was used to define the kinetic mechanism of ATP binding and to determine the elementary rate constants. Using this approach, the kinetic rate constants for ADP, ATPγS, AMPPNP, AMP, adenosine, and inorganic phosphate were also determined at 4 °C in 20 mM Tris·HCl (pH 7.5), 6 mM NaCl, 10% (v/v) glycerol, and 5 mM MgCl2. The kinetics of adenine nucleotide binding to the Rep monomer are similar to those observed with the mant nucleotides under identical experimental conditions (Moore & Lohman, 1994). The kinetic competition data are consistent with the following two-step mechanism for the binding of ATP, ADP, and ATPγS, where P is the Rep monomer and A is theadenine nucleotide: [formula omitted]. In the presence of 5 mM MgCl2, the values of k+1 (∼107M−1 s−1) and k+2 (∼10 s−1) are comparable for each nucleotide, whereas k+2> k−1 for ATP and ATPγS while for ADP k+2 ≪ k−1; hence, differences in the overall equilibrium binding affinities of these nucleotides are primarily due to changes in k−1. This reaction represents the minimal mechanism for the binding of ATP to Rep in the absence of Mg2+, where the four rate constants are similar to those observed with mantATP under the same conditions. Kinetic competition experiments with imidoadenosine 5′-triphosphate (AMPPNP) indicate the presence of an additional slow step to form a third species, (P‒A)**; thus, the binding kinetics of AMPPNP are qualitatively different from those of ATP. The slow apparent binding of AMPPNP to Rep is the consequence of a high value of k−1 rather than a low value of k+1.In contrast, the kinetics of AMP and adenosine (Ado) binding can be described by a single-step mechanism. The overall apparent affinities of Rep for ATP, ADP, AMP, and Ado are ∼108, 106, 104, and 102 M−1, respectively. We have also examined the effects of a variety of salts on the kinetics of mantATP binding to the Rep monomer. The major effect of thesesalts is an apparent reduction in the association rate constant, k+1 [effects decrease in the order (NH4)2SO4 > Na2SO4 > NaPi≫ NH4CI > NaCl], whereas the unimolecular rate constants remain unchanged. Specific effects of both anions and cations are observed, reflecting direct binding of both anions and cations to the Rep monomer, although multivalent anions are most effective. These results are consistent with the competitive binding of anions to the ATP binding site of Rep. Qualitatively similar results are obtained with NaCl and NH4Cl, although at significantly higher concentrations (20‒50 mM), indicating a lower affinity of Rep for Cl, relative to phosphate and sulfate.

Original languageEnglish
Pages (from-to)14565-14578
Number of pages14
Issue number48
StatePublished - Dec 1 1994


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