Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme

Several small ribozymes carry out self-cleavage at a specific phosphodiester bond to yield 2',3'-cyclic phosphate and 5'-hydroxyl termini. Prior mechanistic and structural studies on the HDV ribozymes led to the proposal that the pK(a) of C75 is shifted toward neutrality, making it an effective general acid. Recent mechanistic studies on the hairpin ribozyme have led to models in which protonation of G8 is required for phosphodiester cleavage, either for general acid catalysis or for electrostatic stabilization. Inspection of recent crystal structures of the hairpin ribozyme, including a complex with a vanadate transition state mimic, suggests an alternative model involving general acid-base catalysis with G8 serving as the general base and A38 as the general acid. This model is consistent with the literature on the hairpin ribozyme, including pH-rate profiles of wild-type and mutant ribozymes and solvent isotope effects. General mechanistic considerations for RNA catalysis suggest that the penalty for having general acids and bases with pK(a)s removed from neutrality is not as severe as expected. These considerations suggest that general acid-base catalysis may be a common mechanistic strategy of RNA enzymes.     

Proton transfer dynamics of GART: the pH-dependent catalytic mechanism examined by electrostatic calculations

The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pK(a) of approximately 8. Experimental studies of pH-rate profiles of wild-type and site-directed mutants of GART have led to the proposal that His108, Asp144, and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pK(a) of 9.7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)s of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pK(a) calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, catalysis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histidine, and a low pK(a) for GAR(NH(2)) was calculated, indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART deduced from the experimental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static time-averaged crystallographic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH(2)) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pK(a) of GAR(NH(2)) modeled from the experimental data using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.     

Role of an active site adenine in hairpin ribozyme catalysis

The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.     

An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method

We describe a chemical coupling procedure that allows joining of two RNAs, one of which contains a site-specific base analog substitution, in the absence of divalent ions. This method allows incorporation of nucleotide analogs at specific positions even into large, cis-cleaving ribozymes. Using this method we have studied the effects of substitution of G638 in the cleavage site loop of the VS ribozyme with a variety of purine analogs having different functional groups and pK(a) values. Cleavage rate versus pH profiles combined with kinetic solvent isotope experiments indicate an important role for G638 in proton transfer during the rate-limiting step of the cis-cleavage reaction.     

Activation of the proton transfer pathway in catalysis by iron superoxide dismutase

Catalysis by Escherichia coli and Porphyromonas gingivalis iron superoxide dismutase was activated by addition of primary amines, as measured by pulse radiolysis and stopped-flow spectrophotometry. This activation was saturable for most amines investigated, and a free energy plot of the apparent second-order rate constant of activation was linear as a function of the pK(a) of the amine, indicating activation by proton transfer. Amines provide an alternate rather than the only pathway for proton transfer, and catalysis was appreciable in the absence of amines. Solvent hydrogen isotope effects were near unity for amine activation, which is consistent with rate-contributing proton transfer if the pK(a) of the proton acceptor on the enzyme is not in the region of the pK(a) values of the amines studied, from 7.8 to 10.6. The activation of catalysis by these amines was uncompetitive with respect to superoxide, interpreted as proton transfer in a ternary complex of amine with the enzyme-bound peroxide dianion.     

Determination of proton transfer rates by chemical rescue: application to bacterial reaction centers

The bacterial reaction center (RC) converts light into chemical energy through the reduction of an internal quinone molecule Q(B) to Q(B)H(2). In the native RC, proton transfer is coupled to electron transfer and is not rate-controlling. Consequently, proton transfer is not directly observable, and its rate was unknown. In this work, we present a method for making proton transfer rate-controlling, which enabled us to determine its rate. The imidazole groups of the His-H126 and His-H128 proton donors, located at the entrance of the transfer pathways, were removed by site-directed mutagenesis (His --> Ala). This resulted in a reduction in the observed proton-coupled electron transfer rate [(Q(A)(-)(*)Q(B))Glu(-) + H(+) --> (Q(A)Q(B)(-)(*))GluH], which became rate-controlled by proton uptake to Glu-L212 [Adelroth, P., et al. (2001) Biochemistry 40, 14538-14546]. The proton uptake rate was enhanced (rescued) in a controlled fashion by the addition of imidazole or other amine-containing acids. From the dependence of the observed rate on acid concentration, an apparent second-order rate constant k((2)) for the "rescue" of the rate was determined. k((2)) is a function of the proton transfer rate and the binding of the acid. The dependence of k((2)) on the acid pK(a) (i.e., the proton driving force) was measured over 9 pK(a) units, resulting in a Br?nsted plot that was characteristic of general acid catalysis. The results were fitted to a model that includes the binding (facilitated by electrostatic attraction) of the cationic acid to the RC surface, proton transfer to an intermediate proton acceptor group, and subsequent proton transfer to Glu-L212. A proton transfer rate constant of approximately 10(5) s(-)(1) was determined for transfer from the bound imidazole group to Glu-L212 (over a distance of approximately 20 A). The same method was used to determine a proton transfer rate constant of 2 x 10(4) s(-)(1) for transfer to Q(B)(-)(*). The relatively fast proton transfer rates are explained by the presence of an intermediate acceptor group that breaks the process into sequential proton transfer steps over shorter distances. This study illustrates an approach that could be generally applied to obtain information about the individual rates and energies for proton transfer processes, as well as the pK(a)s of transfer components, in a variety of proton translocating systems.     

The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad.

General-base catalysis in the active site of serine proteases is carried out by the imidazole side chain of a histidine. During formation of the transition state, an adjacent carboxylic acid group stabilizes the positive charge that forms on the general-base catalyst and as a result contributes several orders of magnitude to the catalytic efficiency of these enzymes. In the recently discovered family of self-cleaving proteins exemplified by the LexA repressor of Escherichia coli, instead of the imidazole of a histidine, the active-site general-base catalyst was found to be the epsilon-amino of a lysine. The considerably higher capacity of the lysine side chain for proton acceptance raises interesting questions concerning the role of electrostatic interactions in the mechanism of proton transfer by this highly basic group. The negative charge elimination studies described here and their effects on the kmax and pK of LexA self-cleavage are consistent with a model in which electrostatic interactions between an acidic side chain and the general-base catalyst form a barrier to proton transfer. The implications are that the epsilon-amino group, unlike the imidazole group, is capable of effecting proton transfer without the intervention of a countercharge.     

Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue

The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state.     

Simulating electrostatic energies in proteins: perspectives and some recent studies of pKas, redox, and other crucial functional properties

Electrostatic energies provide what is arguably the most effective tool for structure-function correlation of biological molecules. Here, we provide an overview of the current state-of-the-art simulations of electrostatic energies in macromolecules, emphasizing the microscopic perspective but also relating it to macroscopic approaches. We comment on the convergence issue and other problems of the microscopic models and the ways of keeping the microscopic physics while moving to semi-macroscopic directions. We discuss the nature of the protein dielectric "constants" reiterating our long-standing point that the dielectric "constants" in semi-macroscopic models depend on the definition and the specific treatment. The advances and the challenges in the field are illustrated considering different functional properties including pK(a)'s, redox potentials, ion and proton channels, enzyme catalysis, ligand binding, and protein stability. We emphasize the microscopic overcharging approach for studying pK(a) 's of internal groups in proteins and give a demonstration of power of this approach. We also emphasize recent advances in coarse grained models with a physically based electrostatic treatment and provide some examples including further directions in treating voltage activated ion channels.     

Protonation states and pH titration in the photocycle of photoactive yellow protein

Photoactive yellow protein (PYP) undergoes a light-driven cycle of color and protonation states that is part of a mechanism of bacterial phototaxis. This article concerns functionally important protonation states of PYP and the interactions that stabilize them, and changes in the protonation state during the photocycle. In particular, the chromophore pK(a) is known to be shifted down so that the chromophore is negatively charged in the ground state (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a). The photocycle involves changes of one or both of these protonation states. Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I(2)) intermediate state. Calculations allowing multiple H-bonding arrangements around the chromophore also have been carried out. In addition, ground-state pK(a) values of the chromophore have been measured by UV-visible spectroscopy for the wild-type and the same three mutants. Because of the unusual protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calculations of electrostatic interactions in proteins. Good agreement between experiment and theory can be obtained for the ground state provided the protein interior is assumed to have a relatively low dielectric constant, but only partial agreement between theory and experiment is obtained for the bleached state. We also present a reinterpretation of previously published data on the pH-dependence of the recovery of the ground state from the bleached state. The new analysis implies a pK(a) value of 6.37 for Glu46 in the bleached state, which is consistent with other available experimental data, including data that only became available after this analysis. The new analysis suggests that signal transduction is modulated by the titration properties of the bleached state, which are in turn determined by electrostatic interactions. Overall, the results of this study provide a quantitative picture of the interactions responsible for the unusual protonation states of the chromophore and Glu46, and of protonation changes upon bleaching.     

The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK(a) for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.     

Calculation of pKas in RNA: on the structural origins and functional roles of protonated nucleotides

pK(a) calculations based on the Poisson-Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK(a) shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK(a) shifts of nucleotides in RNA structures using numerical solutions to the Poisson-Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK(a)s of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK(a)s: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions.     

Mechanisms of RNA catalysis

Ribozymes are RNA molecules that act as chemical catalysts. In contemporary cells, most known ribozymes carry out phosphoryl transfer reactions. The nucleolytic ribozymes comprise a class of five structurally-distinct species that bring about site-specific cleavage by nucleophilic attack of the 2'-O on the adjacent 3'-P to form a cyclic 2',3'-phosphate. In general, they will also catalyse the reverse reaction. As a class, all these ribozymes appear to use general acid-base catalysis to accelerate these reactions by about a million-fold. In the Varkud satellite ribozyme, we have shown that the cleavage reaction is catalysed by guanine and adenine nucleobases acting as general base and acid, respectively. The hairpin ribozyme most probably uses a closely similar mechanism. Guanine nucleobases appear to be a common choice of general base, but the general acid is more variable. By contrast, the larger ribozymes such as the self-splicing introns and RNase P act as metalloenzymes.     

Characterisation of an ionisable group involved in binding and catalysis by sialidase from influenza virus.

The effect of pH on the kinetics of sialidase purified from influenza virus (A/Tokyo/3/67, H2N2) was investigated. A pK of 9.0 for inhibition of the enzyme by three competitive inhibitors, due to an ionisable group in the active site, was observed. A similar pK was observed for V/Km for the fluorogenic substrate 2-(4-methylumbelliferyl)-N-acetyl-alpha-D-neuraminic acid. However, the shape of the V/Km profile indicates that this substrate is sticky. Solvent perturbation experiments indicated that the observed ionisable active site group is likely to be a cationic amino acid. The results provide evidence against the hypothesis that Glu 276 acts as a proton donor in the enzyme reaction and supports the proposal of a role for one of the active site cationic amino acids in binding and catalysis.     

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.     

Proton transfer at carbon

The viability of living systems requires that C--H bonds of biological molecules be stable in water, but that there also be a mechanism for shortening the timescale for their heterolytic cleavage through enzymatic catalysis of a variety of catabolic and metabolic reactions. An understanding of the mechanism of enzymatic catalysis of proton transfer at carbon requires the integration of results of studies to determine the structure of the enzyme-substrate complex with model studies on the mechanism for the non-enzymatic reaction in water, and the effect of the local protein environment on the stability of the transition state for this reaction. A common theme is the importance of electrostatic interactions in providing stabilization of bound carbanion intermediates of enzyme-catalyzed proton-transfer reactions.     

Strain and catalysis in aspartate aminotransferase

The notion of "ground-state destabilization" has been well documented in enzymology. It is the unfavourable interaction (strain) in the enzyme-substrate complex, and increases the k(cat) value without changing the k(cat)/K(m) value. During the course of the investigation on the reaction mechanism of aspartate aminotransferase (AAT), we found another type of strain that is crucial for catalysis: the strain of the distorted internal aldimine in the unliganded enzyme. This strain raises the energy level of the starting state (E+S), thereby reducing the energy gap between E+S and ES(++) and increasing the k(cat)/K(m) value. Further analysis on the reaction intermediates showed that the Michaelis complex of AAT with aspartate contains strain energy due to an unfavourable interaction between the main chain carbonyl oxygen and the Tyr225-aldimine hydrogen-bonding network. This belongs to the classical type of strain. In each case, the strain is reflected in the pK(a) value of the internal aldimine. In the historical explanation of the reaction mechanism of AAT, the shifts in the aldimine pK(a) have been considered to be the driving forces for the proton transfer during catalysis. However, the above findings indicate that the true driving forces are the strain energy inherent to the respective intermediates. We describe here how these strain energies are generated and are used for catalysis, and show that variations in the aldimine pK(a) during catalysis are no more than phenomenological results of adjusting the energy levels of the reaction intermediates for efficient catalysis.     

Titration behavior of residues at the entrance of the D-pathway of cytochrome c oxidase from paracoccus denitrificans investigated by continuum electrostatic calculations

Continuum electrostatic calculations were employed to investigate the titration curves of the fully oxidized state of wild type and several variants of cytochrome c oxidase from Paracoccus denitrificans (N131D, N131C, N131V, and D124N) for different values of the dielectric constant of the protein. The effects of the mutations at the entrance of the D-proton transfer pathway were found to be quite localized to their immediate surroundings. The results can be well interpreted in the light of the available biochemical and structural data and help understanding the effects of mutations on proton conductivity. The mutations of aspartic acid Asp-I-124 to a neutral residue resulted in a decreased pK(a) value of His-I-28 suggesting that the mutation of His-I-28 may have a significant influence on the coupling of electron and proton transfer in cytochrome c oxidase. We also investigated the effect of the mutations N131D, N131C, and N131V on the residue Glu-I-278 in terms of its pK(a) value and electrostatic interaction energies.     

Acid-induced exchange of the imino proton in G.C pairs.

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine.