An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis

Background: Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy.Results: Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ionindependent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH.Conclusions: Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metalcation-independent chemical step, such as breaking the 5′ oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.     

Fast cleavage kinetics of a natural hammerhead ribozyme

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.     

Mechanistic characterization of the HDV genomic ribozyme: solvent isotope effects and proton inventories in the absence of divalent metal ions support C75 as the general acid

The hepatitis delta virus (HDV) ribozyme uses the nucleobase C75 and a hydrated Mg(2+) ion as the general acid-base catalysts in phosphodiester bond cleavage at physiological salt. A mechanistic framework has been advanced that involves one Mg(2+)-independent and two Mg(2+)-dependent channels. The rate-pH profile for wild-type (WT) ribozyme in the Mg(2+)-free channel is inverted relative to the fully Mg(2+)-dependent channel, with each having a near-neutral pKa. Inversion of the rate-pH profile was used as the crux of a mechanistic argument that C75 serves as general acid both in the presence and absence of Mg(2+). However, subsequent studies on a double mutant (DM) ribozyme suggested that the pKa observed for WT in the absence of Mg(2+) arises from ionization of C41, a structural nucleobase. To investigate this further, we acquired rate-pH/pD profiles and proton inventories for WT and DM in the absence of Mg(2+). Corrections were made for effects of ionic strength on hydrogen ion activity and pH meter readings. Results are accommodated by a model wherein the Mg(2+)-free pKa observed for WT arises from ionization of C75, and DM reactivity is compromised by protonation of C41. The Br?nsted base appears to be water or hydroxide ion depending on pH. The observed pKa's are related to salt-dependent pH titrations of a model oligonucleotide, as well as electrostatic calculations, which support the local environment for C75 in the absence of Mg(2+) being similar to that in the presence of Mg(2+) and impervious to bulk ions. Accordingly, the catalytic role of C75 as the general acid does not appear to depend on divalent ions or the identity of the Br?nsted base.     

Direct measurement of a pK(a) near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography

The hepatitis delta virus (HDV) ribozyme uses a cytosine to facilitate general acid-base catalysis. Biochemical studies suggest that C75 has a pKa perturbed to near neutrality. To measure this pKa directly, Raman spectra were recorded on single ribozyme crystals using a Raman microscope. A spectral feature arising from a single neutral cytosine was identified at 1528 cm(-1). At low pH, this mode was replaced with a new spectral feature. Monitoring these features as a function of pH revealed pKa values for the cytosine that couple anticooperatively with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These pKa values agree well with those obtained from ribozyme activity experiments in solution. To correlate the observed pKa with a specific nucleotide, crystals of C75U, which is catalytically inactive, were examined. The Raman difference spectra show that this mutation does not affect the conformation of the ribozyme. However, crystals of C75U did not produce a signal from a protonatable cytosine, providing strong evidence that protonation of C75 is being monitored in the wild-type ribozyme. These studies provide the first direct physical measurement of a pKa near neutrality for a catalytic residue in a ribozyme and show that ribozymes, like their protein enzyme counterparts, can optimize the pKa of their side chains for proton transfer.     

Catalysis of hydrolysis and aminolysis of non-classical beta-lactam antibiotics by metal ions and metal chelates.

The Zn(2+)-tris (hydroxymethyl)aminomethane (Tris) system has a great catalytic effect on the hydrolysis and aminolysis of some beta-lactam antibiotics. In order to ascertain the mechanism of this catalysis we have analysed the effects of the beta-lactam antibiotic structure. First we studied the kinetics of the decomposition of imipenem, SCH 29482, aztreonam and nocardicin A in aqueous solution of Tris at 35.0 degrees C, 0.5 mol.dm-3 ionic strength and in the presence of metal ions (Zn2+, Cd2+, Co2+, Cu2+, Ni2+ and Mn2+). From these studies, we conclude that Tris and metal ions (in separate solutions) exert a great catalytic effect on the hydrolysis of imipenem and SCH 29482. We suggest that in metal ion solutions a 1:1 complex is formed between the metal ion and beta-lactam antibiotic, which is attacked by hydroxide ions. Studies of the degradation of the antibiotics studied in solutions of Tris and metal ions together indicate that the systems Cd(2+)-Tris and Zn(2+)-Tris have a great catalytic effect on the hydrolysis and aminolysis of imipenem and SCH 29482. We suggest that this catalysis takes place via a ternary complex in which the metal ion plays a double role by (a) placing the antibiotic and the Tris in the right position for the reaction and (b) lowering the pKa of the hydroxide group of Tris, which is coordinated with the metal ion, generating a strong nucleophile.     

General base catalysis for cleavage by the active-site cytosine of the hepatitis delta virus ribozyme: QM/MM calculations establish chemical feasibility

The hepatitis delta virus (HDV) ribozyme is an RNA motif embedded in human pathogenic HDV RNA. Previous experimental studies have established that the active-site nucleotide C75 is essential for self-cleavage of the ribozyme, although its exact catalytic role in the process remains debated. Structural data from X-ray crystallography generally indicate that C75 acts as the general base that initiates catalysis by deprotonating the 2'-OH nucleophile at the cleavage site, while a hydrated magnesium ion likely protonates the 5'-oxygen leaving group. In contrast, some mechanistic studies support the role of C75 acting as general acid and thus being protonated before the reaction. We report combined quantum chemical/molecular mechanical calculations for the C75 general base pathway, utilizing the available structural data for the wild type HDV genomic ribozyme as a starting point. Several starting configurations differing in magnesium ion placement were considered and both one-dimensional and two-dimensional potential energy surface scans were used to explore plausible reaction paths. Our calculations show that C75 is readily capable of acting as the general base, in concert with the hydrated magnesium ion as the general acid. We identify a most likely position for the magnesium ion, which also suggests it acts as a Lewis acid. The calculated energy barrier of the proposed mechanism, approximately 20 kcal/mol, would lower the reaction barrier by approximately 15 kcal/mol compared with the uncatalyzed reaction and is in good agreement with experimental data.     

A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage

Background: Ribozymes are biological catalysts that promote the hydrolysis and transesterification of phosphate diesters of RNA. They typically require divalent magnesium ions for activation, although it has proven difficult to differentiate structural from catalytic roles for the magnesium ions and to identify the molecular mechanism of catalysis. Direct inner-sphere coordination is usually invoked in the catalytic step, although there is no evidence to support the generality of such a pathway for all ribozymes.Results: We studied the catalytic pathway for the hairpin class of ribozyme. The substitutionally inert transition metal complex cobalt hexaammine [Co(NH₃)₆³⁺) was shown to be as active as Mg²⁺(aq) in promoting hairpin ribozyme activity, demonstrating that inner-sphere pathways are not used by this class of ribozyme. These results were confirmed by studies with R_P- and S_P-phosphorothioate substrate analogs which show a similar reactivity to that of the native substrate towards the magnesium-activated ribozyme. Monovalent cations enhance the activity of Co(NH₃)₆³⁺-promoted reactions, but inhibit Mg²⁺-activated catalysis, demonstrating a requirement for hydrated cations at several key sites in the ribozyme.Conclusions: These results provide clear support for a model of RNA catalysis that does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule. A mechanism in which catalysis is carried out by functional groups on the RNA ribozyme itself is possible; such functional groups are likely to have pK_a values that are appropriate for carrying out this catalysis. The metal cofactor would then serve to define the architecture of the catalytic pocket and contribute to the stabilization of transient species, as has been described earlier. Hydrolytic pathways in nucleic acid reactions are apparently more diverse than was previously thought, and the hairpin ribozyme falls into a mechanistically distinct class from the Tetrahymena and the hammerhead ribozymes.     

Acid-base and metal-ion binding properties of the RNA dinucleotide uridylyl-(5'-->3')-[5']uridylate (pUpU3-)

It is well known that Mg2+ and other divalent metal ions bind to the phosphate groups of nucleic acids. Subtle differences in the coordination properties of these metal ions to RNA, especially to ribozymes, determine whether they either promote or inhibit catalytic activity. The ability of metal ions to coordinate simultaneously with two neighboring phosphate groups is important for ribozyme structure and activity. However, such an interaction has not yet been quantified. Here, we have performed potentiometric pH titrations to determine the acidity constants of the protonated dinucleotide H2(pUpU)-, as well as the binding properties of pUpU3- towards Mg2+, Mn2+, Cd2+, Zn2+, and Pb2+. Whereas Mg2+, Mn2+, and Cd2+ only bind to the more basic 5'-terminal phosphate group, Pb2+, and to a certain extent also Zn2+, show a remarkably enhanced stability of the [M(pUpU)]- complex. This can be attributed to the formation of a macrochelate by bridging the two phosphate groups within this dinucleotide by these metal ions. Such a macrochelate is also possible in an oligonucleotide, because the basic structural units are the same, despite the difference in charge. The formation degrees of the macrochelated species of [Zn(pUpU)]- and [Pb(pUpU)]- amount to around 25 and 90 %, respectively. These findings are important in the context of ribozyme and DNAzyme catalysis, and explain, for example, why the leadzyme could be selected in the first place, and why this artificial ribozyme is inhibited by other divalent metal ions, such as Mg2+.     

铜的表面氧在甲醇水蒸气重整反应中的作用

研究了Cu和ZrO₂/Cu模型催化剂的甲醇水蒸气重整制氢的反应性能, 结果表明, 纯铜催化剂的反应初始活性随着还原温度的增加而显著降低, 并且在失活后的催化剂反应体系中通入少量的氧, 可恢复催化剂的活性. 相对于Cu, ZrO₂/Cu催化剂的活性和稳定性显著增加. 催化剂的TPR, XPS以及原位FT-IR表征结果表明, 导致催化剂活性迅速降低的原因为催化剂表面氧物种的逐渐消耗. ZrO₂在反应过程中可以稳定铜表面氧以及Cu^＋物种, 从而显著提高了反应活性和稳定性.     

Density functional theory investigation on the mechanism of the hepatitis delta virus ribozyme

Density functional theory methods have been used to investigate the hepatitis delta virus (HDV) ribozyme and its catalyzed phosphodiester cleavage. In particular, the effects of the environment's polarity and/or specific hydrogen-bond interactions on the proton affinity of the active site cytosine's N3 ring center have been considered. In addition, the basicities of possible hydrated Mg2+ ion species were also examined. The mechanism previously proposed for the HDV ribozyme in which the active site cytosine (C75) is protonated and thus acts as an acid while the Mg2+ species acts as the complementary base was then investigated. The possible role of tautomerization of C75 is also discussed.     

Effect of Ni(ii), Cu(ii) and Zn(ii) association on the keto-enol tautomerism of thymine in the gas phase

The effect of Ni(II), Cu(II) and Zn(II) association on the diketo/keto-enol tautomerism of thymine has been investigated through the use of B3LYP density functional theory calculations. Final energies were obtained at the B3LYP/6-311+G(2df,2p)//B3LYP/6-311+G(d,p) level of theory. Ni(II) and Cu(II) lead to an oxidation of thymine which for Zn(ii) is only partial and catalyze the tautomerization process, this catalytic effect being much larger for Ni(2+) and Zn(2+) than for Cu(2+). One of the most significant consequences of the oxidation of the base is that the calculated BDE's are primarily dictated by the value of the second ionization potential of the metal, and therefore follow the sequence Cu(2+) > Ni(2+) > Zn(2+). Also importantly, metal dication association leads to a stabilization of the keto-enol tautomer, which becomes the most stable form upon interaction with Ni(2+) and Zn(2+). This stabilization enhancement is the consequence of three concomitant factors, namely, (i) a stronger interaction of the metal cation with the carbonyl oxygen, (ii) the interaction of the metal with the dehydrogenated ring nitrogen, (iii) an aromatization of the six-membered ring.     

Probing general base catalysis in the hammerhead ribozyme

Recent structural and computational studies have shed new light on the catalytic mechanism and active site structure of the RNA cleaving hammerhead ribozyme. Consequently, specific ribozyme functional groups have been hypothesized to be directly involved in general/acid base catalysis. In order to test this hypothesis, we have developed an affinity label to identify the functional general base in the S. mansoni hammerhead ribozyme. The ribozyme was reacted with a substrate analogue bearing a 2'-bromoacetamide group in place of the nucleophilic 2'-hydroxyl group which would normally be deprotonated by a general base. The electrophilic 2'-bromoacetamide group is poised to alkylate the general base, which is subsequently identified by footprinting analysis. Herein, we demonstrate alkylation of N1 of G12 in the hammerhead ribozyme in a pH and [Mg(2+)] dependent manner that is consistent with the native cleavage reaction. These results provide substantial evidence that deprotonated N1 of G12 functions directly as a general base in the hammerhead ribozyme; moreover, our experiments provide evidence that the pKa of G12 is perturbed downward in the context of the active site structure. We also observed other pH-independent alkylations, which do not appear to reflect the catalytic mechanism, but offer further insight into ribozyme conformation and structure.     

A structure-based 3D-QSAR study of anthrapyrazole analogues of the anticancer agents losoxantrone and piroxantrone

A series of 13 anthrapyrazole compounds that are analogues of piroxantrone and losoxantrone were synthesized, and their cell growth inhibitory effects, DNA binding, topoisomerase IIalpha mediated (EC 5.99.1.3) cleavage of DNA, and inhibition of DNA topoisomerase IIalpha decatenation catalytic activities were determined. Cell growth inhibitory activity was well-correlated with DNA binding, suggesting that these compounds may act by targeting DNA. However, cell growth inhibition was not well-correlated with the inhibition of topoisomerase IIalpha catalytic activity, suggesting that these anthrapyrazoles did not act solely by inhibiting the catalytic activity of topoisomerase II. Most of the analogues were able to induce DNA cleavage, and thus, it was concluded that they acted, at least in part, as topoisomerase II poisons. Structure-based three-dimensional quantitative structure-activity analyses (3D-QSAR) were carried out on the aligned structures of the anthrapyrazoles docked into DNA using comparative molecular field analysis (CoMFA) and comparative molecular similarity index (CoMSIA) analyses in order to determine the structural features responsible for their activity. Both CoMFA and CoMSIA yielded statistically significant models upon partial least-squares analyses. The 3D-QSAR analyses showed that hydrogen-bond donor interactions and electrostatic interactions with the protonated amino side chains of the anthrapyrazoles led to high cell growth inhibitory activity.     

基于银/铂纳米模拟酶表面修饰的铜离子比色检测方法研究

通过对银/铂纳米簇(Ag/Pt NCs)的表面修饰调控其催化活性,建立了一种高灵敏的比色法检测Cu2+.巯基丙酸能够抑制Ag/Pt NCs的催化活性,而巯基丙酸与Cu2+作用后,将导致上述抑制作用减弱.基于上述原理,通过测量Ag/Pt NCs 催化TMB-H2O2反应产生的显色信号,可以实现Cu2+的比色检测.本方法检测Cu2+的线性范围为10～100 nmol/L,检出限(3σ)为5.0 nmol/L.将本方法应用于实际水样中Cu2+的检测,结果表明,本方法具有操作简单、成本低、灵敏度高、特异性好等优点.     

Synthesis and Enzymic Hydrolysis of Oligoribonucleotides Incorporating 3‐Deazaguanosine: The Importance of the Nitrogen‐3 Atom of Single Conserved Guanosine Residues on the Catalytic Activity of the Hammerhead Ribozyme

Four base‐modified hammerhead ribozyme/substrate complexes were constructed in which single guanosine ( 1 ) residues were replaced by 3‐deazaguanosine ( 2 ) in the positions G₅, G₈, G_L2.1, and G₁₂. The base‐modified ribozyme complexes were prepared by solid‐phase synthesis of oligoribonucleotides employing the novel phosphoramidite 3 derived from 2 . Phosphoramidite 3 carried a phenoxyacetyl group at the amino function and a diphenylcarbamoyl residue at the oxo group of the nucleobase. The 2′‐hydroxy group was blocked with a triisopropylsilyl residue. Kinetic analysis of the phosphodiester hydrolysis showed a moderate decrease of the ribozyme catalytic activity when the residues G₅ or G₈ were replaced by 3‐deazaguanosine and a 200‐fold decrease when G₁₂ was substituted. A 6‐fold catalytic increase occurred when 3‐deazaguanosine was replacing G_L2.1 in the loop region. The data indicate that the N(3) atom of compound 2 , in particular at position G₁₂ is critical for the ribozyme activity.     

Solvent structure and hammerhead ribozyme catalysis

Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.     

Promiscuous catalysis by the tetrahymena group I ribozyme

Catalytic promiscuity, the ability of an enzyme to catalyze alternative reactions, has been suggested to have played an important role in the evolution of new catalytic activities in protein enzymes. Similarly, promiscuous activities may have been advantageous in an earlier RNA world. The Tetrahymena Group I ribozyme naturally catalyzes the site-specific guanosine attack on an anionic phosphate diester and has been shown to also catalyze aminoacyl transfer to water, albeit with a small rate acceleration (<10-fold). This inefficient catalysis could be due to the differences in charge and/or geometry requirements for the two reactions. Herein, we describe a new promiscuous activity of this ribozyme, the site-specific guanosine attack on a neutral phosphonate diester. This alternative substrate lacks the negative charge at the reaction center but, in contrast to the aminoacyl substrate, can undergo nucleophilic attack with the same geometry as the natural substrate. Our results show that the neutral phosphonate reaction is catalyzed about 1 x 106-fold, substantially better than the acyl transfer but far below the normal anionic substrate. We conclude that both charge and geometry are important factors for catalysis of the normal reaction and that promiscuous catalytic activities of ribozymes could have been created or enhanced by reorienting and swapping RNA domains.     

Peptidyl-transferase ribozymes: trans reactions,structural characterization and ribosomal RNA-like features

Background: One of the most significant questions in understanding the origin of life concerns the order of appearance of DNA, RNA and protein during early biological evolution. If an ‘RNA world’ was a precursor to extant life, RNA must be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form amide bonds between RNA and an amino acid or between two amino acids.Results: We characterized peptidyl-transferase reactions catalyzed by two different families of ribozymes that use substrates that mimic A site and P site tRNAs. The family II ribozyme secondary structure was modeled using chemical modification, enzymatic digestion and mutational analysis. Two regions resemble the peptidyl-transferase region of 23S ribosomal RNA in sequence and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular (‘trans’) peptide-bond formation, with the two amino-acid substrates binding through an attached AMP or oligonucleotide moiety.Conclusions: An in vitro-selected ribozyme can catalyze the same type of peptide-bond formation as a ribosome; the ribozyme resembles the ribosome because a very specific RNA structure is required for substrate binding and catalysis, and both amino acids are attached to nucleotides. It is intriguing that, although there are many different possible peptidyl-transferase ribozymes, the sequence and secondary structure of one is strikingly similar to the ‘helical wheel’ portion of 23S rRNA implicated in ribosomal peptidyl-transferase activity.     

Nucleobase participation in ribozyme catalysis

We constructed a modified form of the VS ribozyme containing an imidazole ring in place of adenine at position 756. The novel ribozyme is active in both cleavage and ligation reactions. The reaction is efficient, although relatively slow. The results are consistent with a role for nucleobase catalysis in the catalytic mechanism of this ribozyme.     

Structure, bonding, and catecholase mechanism of copper bispidine complexes

Oxygen activation by copper(I) complexes with tetra- or pentadentate mono- or dinucleating bispidine ligands is known to lead to unusually stable end-on-[{(bispidine)Cu}(2)(O(2))](2+) complexes (bispidines are methyl-2,4-bis(2-pyridin-yl)-3,7-diazabicyclo-[3.3.1]-nonane-9-diol-1,5-dicarboxylates); catecholase activity of these dinuclear Cu(II/I) systems has been demonstrated experimentally, and the mechanism has been thoroughly analyzed. The present density functional theory (DFT) based study provides an analysis of the electronic structure and catalytic activity of [{(bispidine)Cu}(2)(O(2))](2+). As a result of the unique square pyramidal coordination geometry, the d(x(2)-y(2)) ground state leads to an unusual σ/π bonding pattern, responsible for the stability of the peroxo complex and the observed catecholase activity with a unique mechanistic pathway. The oxidation of catechol to ortho-quinone (one molecule per catalytic cycle and concomitant formation of one equivalent of H(2)O(2)) is shown to occur via an associative, stepwise pathway. The unusual stability of the end-on-peroxo-dicopper(II) complex and isomerization to copper(II) complexes with chelating catecholate ligands, which inhibit the catalytic cycle, are shown to be responsible for an only moderate catalytic activity.