12 research outputs found

    Initial Characterization of a Conserved Active Site Residue for the Cdc34 Ubiquitin Conjugating Enzyme

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    Ubiquitin-conjugating enzymes (E2s) covalently modify protein substrates with ubiquitins. The active site cysteine residues on E2s are essential for catalyzing the transfer of ubiquitin from the E2 active site onto the protein substrate, however there is a limited amount of information available concerning additional active site residues for E2s that may also participate in catalysis. Cdc34 is an essential E2 that has merited the lion’s share of attention for biochemical analysis of the E2 family. Previous phylogenetic analysis of Cdc34 amino acid sequences has identified an invariably conserved histidine residue close to the active site cysteine in the primary structure, however whether this residue actually participates in Cdc34 function is unknown. Here we demonstrate that histidine 98 on the human Cdc34 ubiquitin conjugating enzyme is vital to the enzymatic activity of the E2. Recombinant His98Ala mutant Cdc34 was isolated from bacterial cells engineered to express the protein and compared to wild-type Cdc34 through two complementary ubiquitination assays. Substitution of the histidine residue with alanine resulted in a nearly complete loss of function. These results uncover the possible roles that histidine 98 may play during Cdc34 function

    A Faster Triphosphorylation Ribozyme.

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    In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5'-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min-1 under selection conditions (50 mM Tmp, 100 mM MgCl2, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme's structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl2, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min-1

    The NTP binding site of the polymerase ribozyme.

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    Triphosphorylation kinetics of TPR1e.

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    <p><b>(A)</b> At a Tmp concentration of 1 mM, the triphosphorylation kinetics are shown for synthetic seawater at 22°C (black triangles, k<sub>obs</sub> = 0.0014 min<sup>-1</sup>, max = 93%), and at 40°C (empty squares, k<sub>obs</sub> = 0.0039 min<sup>-1</sup>, max = 97%). For comparison, the reaction kinetics are shown for 54 mM MgCl<sub>2</sub> in Tris/HCl pH 8.3 at 22°C (black circles, k<sub>obs</sub> = 0.020 min<sup>-1</sup>, max = 93%). This latter condition lacks all seawater components with exception of Mg<sup>2+</sup>. Error bars are standard deviations from triplicate experiments, and are smaller than the symbols if not visible. Curves are single-exponential fits to the data. <b>(B)</b> Titration of the Tmp concentration in the reaction at 22°C, in synthetic seawater (black triangles) and in 54 mM MgCl<sub>2</sub> with 50 mM Tris/HCl pH 8.3 (black circles). The offset between the linear fits (grey lines) is 15-fold, on average. <b>(C)</b> Titration of sodium chloride into a triphosphorylation ribozyme reaction containing 50 mM Tmp and 140 mM MgCl<sub>2</sub>. The grey line is a single-exponential fit to the data (with offset) and identifies a 1.9-fold reduction in k<sub>obs</sub> at 470 mM [NaCl], the same NaCl concentration as in synthetic seawater (dashed line). Error bars are standard deviations from triplicate experiments, and are smaller than the symbols if not visible.</p

    Determination of optimal TPR1e triphosphorylation conditions.

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    <p><b>(A)</b> Observed triphosphorylation rate as function of the temperature, at 50 mM Tmp, 100 mM MgCl<sub>2</sub>, and 50 mM Tris/HCl pH 8.3 <b>(B)</b> Influence of the trimetaphosphate concentration on the observed reaction kinetics at 40°C, and with an excess of 400 mM MgCl<sub>2</sub> over Tmp. <b>(C)</b> Influence of the free Mg<sup>2+</sup> concentration on the triphosphorylation rate at 40°C and 150 mM Tmp. The free Mg<sup>2+</sup> concentration is the total Mg<sup>2+</sup> concentration minus the concentration of Tmp because each Tmp appears to be coordinated by one Mg<sup>2+</sup> at these concentrations and pH 8.3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142559#pone.0142559.ref016" target="_blank">16</a>]. The grey arrows indicate the optimum condition for each series of experiments. Note that the scale in (A) is different from the scale in (B) and (C). Error bars are standard deviations of triplicate experiments.</p

    Triphosphorylation kinetics of central ribozyme variants in this study.

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    <p>The starting point of the doped selection was the ribozyme TPR1 (empty circles). It has a k<sub>obs</sub> of 0.013 min<sup>-1</sup> under selection conditions (100 mM MgCl<sub>2</sub>, 50 mM trimetaphosphate, 50 mM Tris/HCl pH 8.3). The most efficient isolate from the doped selection was a 16-mutation variant called clone 11 (filled triangles, k<sub>obs</sub> of 0.21 min<sup>-1</sup>). After removal of unnecessary mutations a 5-mutation variant called TPR1-II resulted (open squares, k<sub>obs</sub> of 0.25 min<sup>-1</sup>). Two mutations that arose independently were introduced to yield TPR1e (filled squares), a 7-mutation variant with a k<sub>obs</sub> of 0.31 min<sup>-1</sup>. Lines are single-exponential curve fits to the data. Error bars denote the standard deviations from triplicate experiments.</p