Decapping kinetics under conditions of excess DcpS relative to cap substrate. A, recombinant FLAG-DcpS was overexpressed and purified from HEK293T cells and resolved by SDS-PAGE, followed by Sypro-Ruby staining. B, decapping reactions were carried out using monomer concentrations of 20, 50, 100, and 200 nm DcpS and 10 nm unlabeled cap structure substrate spiked with α-³²P-labeled cap by a rapid quenched-flow instrument at 25 °C. The hydrolysis product and unhydrolyzed substrate were resolved by TLC developed in 0.45 m (NH₄)₂SO₄ and exposed to a phosphor screen and developed on a PhosphorImager (see “Experimental Procedures”). C, the fraction of generated products was quantitated and plotted against the logarithmic value of the reaction time. The data points of each experiment were fit to a single exponential equation (Equation 1) to obtain the psuedo-first order decapping rate constants. The data represented here are an average of two sets of independent experiments carried out on two different days. The error bars indicate the data range for each time point of the experiment. D, the rate constants derived from C were plotted against DcpS concentrations. The slope of this linear plot gave the biomolecular rate constant for the cap hydrolysis. This value ranged between 2.3 ± 0.1 × 10⁶ and 6.8 ± 0.6 × 10⁶ m^–1 s^–1 based on four different data sets obtained through independent experimentation. E, the amount of m⁷Gp product generated was calculated from C and plotted against DcpS concentrations. Even under the lowest enzyme concentration tested here (20 nm), the reaction went to completion, indicating that the enzyme exhibits a very tight binding for the substrate and has an equilibrium dissociation constant (K_d) of less than 20 nm.

Decapping kinetics under conditions of excess substrate relative to DcpS. A, 100 nm monomer concentration of DcpS was reacted with 200, 400, 800, and 1600 nm cap substrate. The fraction of decapping product generated was plotted against the reaction time. The solid lines represent data points of each experiment fit to a single exponential equation (Equation 1). The data represented here are an average of three sets of independent experiments carried out on different days. The error bars indicate the data range for each time point of the experiment. B, the psuedo-first order decapping rate constants (initial rate (nm s^–1)/[DcpS]) were plotted against DcpS concentrations. The reaction rates appear to have saturated in the range of substrate concentrations used and imply a maximum rate constant of 0.0925 ± 0.003 s^–1.

Trapping of cap substrate at the HIT mutant site of DcpS^WT/HIT prevents hydrolysis at the WT active site. A, a schematic representation of the double-tagged DcpS^WT/HIT heterodimer is shown. The HIT site binds cap structure but is unable to hydrolyze it (24). B, recombinant DcpS^WT/HIT was overexpressed and purified from BL21-CodonPlus(DE3)-RIPL bacterial cells and resolved by SDS-PAGE, followed by Sypro-Ruby staining. Migration of the DcpS^WT/HIT heterodimer is indicated as is the copurifying bacterial GroEL, whose identity was determined by mass spectrometry. C, an electrophoretic mobility shift assay was used to test the ability of DcpS^WT/HIT heterodimer to bind ³²P-labeled cap analog. The DcpS^WT/HIT heterodimer containing two different tags that were used to selectively purify the protein possesses a FLAG-tagged wild type protomer and a His-tagged HIT mutant protomer (lane 3). The His-tagged wild type homodimer was used in lane 2, and His-tagged HIT mutant homodimer was used in lane 4. The electrophoretic mobility shift assay reactions were carried out with 1 μg of the indicated proteins and resolved on a 5.6% polyacrylamide gel. D, the rapid quenched-flow decapping assays of DcpS^WT/HIT were carried out under single and multiple turnover conditions and resolved by TLC. Single turnover conditions of 100 nm monomer concentration of enzyme and 10 nm substrate are denoted by the circles. ×, multiple turnover conditions of 100 nm monomer concentration of enzyme and 800 nm substrate. The fractions of generated decapping product in each set of experiments were quantitated and plotted against the reaction times. The data points of the single turnover condition were fit to a biphasic, double exponential curve (Equation 2). The data represented here are an average of two sets of independent experiments carried out on different days. The error bars indicate the data range for each time point of the experiment. The maximal decapping rate of the multiple turnover experiment is lower than 2%.

Both DcpS^WT/WT homodimer and DcpS^WT/BC heterodimer display decreased decapping rates under multiple turnover conditions. A, schematic representations of the double-tagged DcpS^WT/WT homodimer and DcpS^WT/BC heterodimer are shown. With the domain-swapped nature of DcpS, DcpS^WT/BC contains a mutation in each protomer, but upon heterodimerization the two mutations assemble at the same binding site, rendering this site catalytically compromised. B, recombinant DcpS^WT/WT and DcpS^WT/BC were overexpressed and purified from BL21-CodonPlus(DE3)-RIPL bacterial cells and resolved by SDS-PAGE and visualized by Sypro-Ruby staining. C, the DcpS^BC/BC homodimer has no detectable decapping activity with the decapping conditions employed. An in vitro decapping assay was carried out with the indicated monomer concentrations of DcpS^BC/BC homodimer and 200 nm unlabeled cap structure spiked with ³²P-labeled cap structure. The reactions were incubated for 30 s at room temperature and terminated by 1.7 n formic acid. The hydrolyzed product m⁷Gp and unhydrolyzed cap structure substrate were resolved by TLC and exposed to the phosphor screen and developed on a PhosphorImager. D, rapid quenched-flow decapping assays of DcpS^WT/BC carried out under single turnover and multiple turnover conditions. A 100 nm monomer concentration of DcpS^WT/BC was used with 10 nm cap substrate spiked with ³²P-labeled cap substrate under single turnover conditions. 100 nm monomer concentration of DcpS^WT/BC was used with 800 nm cap substrate spiked with ³²P-labeled cap substrate under multiple turnover conditions. The fraction of decapping product generated in each experiment was carried out as duplicates on two different days. The averages of both of these data sets have been quantitated and plotted against the reaction times, with the error bars indicating the data range obtained with each set. The data points for each set of experiments were fit to a single exponential equation (Equation 1) to obtain the rate constant.

Models of decapping mechanism under single (low substrate/enzyme ratio) and multiple (high substrate/enzyme ratio) turnover conditions. A, decapping model under conditions when substrate concentration is less than enzyme active sites displays no cooperativity between the two protomers. The ligand-free DcpS dimer exhibits a symmetric conformation or open conformation. Upon binding to the cap substrate at one binding site, the N terminus at this site closes at a fast rate for substrate cleavage (closed conformation), followed by releasing of the decapping product m⁷Gp and ppN to complete a catalytic cycle. Only one protomer is used per cycle; therefore, there is no allosteric communication between the first and second protomers. B, DcpS hydrolysis of cap substrate displays negative cooperativity under conditions where substrate concentration is in excess of enzyme active sites. Under high substrate conditions, two substrates are bound to the dimer, and the conformational change involved in the closing of the N terminus on one of the substrates is either an unfavorable step or a slow limiting step. The rigidity of the domain-swapped region restrains the N terminus of the second protomer to stay in an open position. After the substrate bound at the first site is hydrolyzed, the decapping products are released, and the N terminus of the second site closes at the same slow rate for hydrolysis, followed by product release to complete the catalytic cycle.