Mononuclear and dinuclear mechanisms for catalysis of phosphodiester cleavage by alkaline earth metal ions in aqueous solution.

In biological systems, enzymes often use metal ions, especially Mg(2+), to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg(2+) and Ca(2+) catalyzed hydrolysis of the model phosphodiester thymidine-5'-p-nitrophenyl phosphate (T5PNP). At 25 degrees C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (k(OH)) of 4.1x10(-4)M(-1)s(-1) and 3.7x10(-4)M(-1)s(-1) in the presence of 0.30M Mg(2+) and Ca(2+), respectively, compared to 8.3x10(-7)M(-1)s(-1) in the absence of divalent metal ion. Examining the dependence of k(OH) on [M(2+)] at 50 degrees C indicates different kinetic mechanisms with Mg(2+) utilizing a single ion mechanism and Ca(2+) operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k(OH) values reveals a single Mg(2+) catalyzes the reaction by 1800-fold whereas a single Ca(2+) ion catalyzes the reaction by only 90-fold. The second Ca(2+) provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg(2+) and Ca(2+).

Experimental analyses of the chemical dynamics of ribozyme catalysis.

Most ribozymes in Nature catalyze alcoholysis or hydrolysis of RNA phosphodiester bonds. Studies of the corresponding non-enzymatic reactions reveal a complex mechanistic landscape allowing for a variety of transition states and both concerted and stepwise mechanisms. High-resolution structures, incisive biochemical studies and computer simulations are providing glimpses into how ribozyme catalyzed reactions traverse this landscape. However, direct experimental tests of mechanistic detail at the chemical level are not easily achieved. Kinetic isotope effects (KIEs) probe directly the differences in the vibrational 'environment' of the atoms undergoing chemical transformation on going from the ground state to the transition state. Thus, KIEs can in principle provide direct information about transition state bonding and so may be instrumental in evaluating possible transition states for ribozyme catalyzed reactions. Understanding charge distribution in the transition state may help resolve how rate acceleration is accomplished and perhaps the similarities and differences in how RNA and protein active sites operate. Several barriers to successful application of KIE analysis to ribozymes have recently been overcome, and new chemical details are beginning to emerge.

Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide 18O/16O measurements.

Precise and accurate measurements of isotopologue distributions (IDs) in biological molecules are needed for determination of isotope effects, quantitation by isotope dilution, and quantification of isotope tracers employed in both metabolic and biophysical studies. While single ion monitoring (SIM) yields significantly greater sensitivity and signal/noise than profile-mode acquisitions, we show that small changes in the SIM window width and/or center can alter experimentally determined isotope ratios by up to 5%, resulting in significant inaccuracies. This inaccuracy is attributed to mass granularity, the differential distribution of digital data points across the m/z ranges sampled by SIM. Acquiring data in the profile mode and fitting the data to an equation describing a series of equally spaced and identically shaped peaks eliminates the inaccuracies associated with mass granularity with minimal loss of precision. Additionally a method of using the complete ID profile data that inherently corrects for "spillover" and for the natural-abundance ID has been used to determine 18O/16O ratios for 5',3'-guanosine bis-[18O1]phosphate and TM[18O1]P with precisions of approximately 0.005. The analysis protocol is also applied to quadrupole time-of-flight tandem mass spectrometry using [2-(18)O] arabinouridine and 3'-UM[18O1]P which enhances signal/noise and minimizes concerns for background contamination.

Analysis of solvent nucleophile isotope effects: evidence for concerted mechanisms and nucleophilic activation by metal coordination in nonenzymatic and ribozyme-catalyzed phosphodiester hydrolysis.

Heavy atom isotope effects are a valuable tool for probing chemical and enzymatic reaction mechanisms; yet, they are not widely applied to examine mechanisms of nucleophilic activation. We developed approaches for analyzing solvent (18)O nucleophile isotope effects ((18)k(nuc)) that allow, for the first time, their application to hydrolysis reactions of nucleotides and nucleic acids. Here, we report (18)k(nuc) for phosphodiester hydrolysis catalyzed by Mg(2+) and by the Mg(2+)-dependent RNase P ribozyme and deamination by the Zn(2+)-dependent protein enzyme adenosine deaminase (ADA). Because ADA incorporates a single solvent molecule into the product inosine, this reaction can be used to monitor solvent (18)O/(16)O ratios in complex reaction mixtures. This approach, combined with new methods for analysis of isotope ratios of nucleotide phosphates by whole molecule mass spectrometry, permitted determination of (18)k(nuc) for hydrolysis of thymidine 5'-p-nitrophenyl phosphate and RNA cleavage by the RNase P ribozyme. For ADA, an inverse (18)k(nuc) of 0.986 +/- 0.001 is observed, reflecting coordination of the nucleophile by an active site Zn(2+) ion and a stepwise mechanism. In contrast, the observed (18)k(nuc) for phosphodiester reactions were normal: 1.027 +/- 0.013 and 1.030 +/- 0.012 for the Mg(2+)- and ribozyme-catalyzed reactions, respectively. Such normal effects indicate that nucleophilic attack occurs in the rate-limiting step for these reactions, consistent with concerted mechanisms. However, these magnitudes are significantly less than the (18)k(nuc) observed for nucleophilic attack by hydroxide (1.068 +/- 0.007), indicating a "stiffer" bonding environment for the nucleophile in the transition state. Kinetic analysis of the Mg(2+)-catalyzed reaction indicates that a Mg(2+)-hydroxide complex is the catalytic species; thus, the lower (18)k(nuc), in large part, reflects direct metal ion coordination of the nucleophilic oxygen. A similar value for the RNase P ribozyme catalyzed reaction provides support for nucleophilic activation by metal ion catalysis.

Understanding the transition states of phosphodiester bond cleavage: insights from heavy atom isotope effects.

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.

Evidence for direct attack by hydroxide in phosphodiester hydrolysis.

Phosphodiester hydrolysis has been the subject of intense study due to its importance in biology. Despite the numerous significant analyses of phosphodiester cleavage mechansim, comparatively little is known about the nucleophiles in these reactions. To determine whether hydroxide acts as a nucleophile or a general base in the hydrolysis of thymidine-5'-p-nitrophenyl phosphate,we determined solvent deuterium isotope effects (D2Ok), ionic strength effects, and 18O isotope effects on the solvent nucleophile (18knuc). The D2Ok for hydroxide-catalyzed phosphodiester hydrolysis is slightly inverse (0.9 +/- 0.1), suggesting that a proton transfer does not occur in the transition state. A significant alpha effect is observed with hydroperoxide, demonstrating that oxyanions can act as nucleophiles in the reaction. Additionally, the ionic strength dependencies of hydroxide and hydroperoxide catalysis are indistinguishable, suggesting that they act by the same mechanism. Finally, the 18knuc for the hydroxide-catalyzed reaction is 1.068 +/- 0.007, well in excess of the equilibrium 18O isotope effect between water and hydroxide (1.040 +/- 0.003). Together, the data are most consistent with direct nucleophilic attack by hydroxide. From the observed 18knuc and the known equilibrium component, the kinetic component of the isotope effect was calculated to be 1.027 +/- 0.010. This large kinetic component suggests that little bond order to the nucleophile occurs in the transition state.

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis.

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex (K(d)=5.4(+/-0.6) nM), and the rate constant for phosphodiester bond cleavage (k(c)=1.160(+/-0.001) min(-1), pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5' phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k(c), fits best to a model for two or more titratable groups with pK(a) of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k(c) is dependent on the pK(a) value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(+/-0.1)x10(8) M(-1) s(-1), while the rate constant of the second phase was concentration independent (6.4(+/-0.8) s(-1); pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.