The role of the cleavage site 2'-hydroxyl in the Tetrahymena group I ribozyme reaction

BACKGROUND: The 2'-hydroxyl of U preceding the cleavage site, U(-1), in the Tetrahymena ribozyme reaction contributes 10(3)-fold to catalysis relative to a 2'-hydrogen atom. Previously proposed models for the catalytic role of this 2'-OH involve coordination of a catalytic metal ion and hydrogen-bond donation to the 3'-bridging oxygen. An additional model, hydrogen-bond donation by the 2'-OH to a nonbridging reactive phosphoryl oxygen, is also consistent with previous results. We have tested these models using atomic-level substrate modifications and kinetic and thermodynamic analyses. RESULTS: Replacing the 2'-OH with -NH(3)(+) increases the reaction rate approximately 60-fold, despite the absence of lone-pair electrons on the 2'-NH(3)(+) group to coordinate a metal ion. Binding and reaction of a modified oligonucleotide substrate with 2'-NH(2) at U(-1) are unaffected by soft-metal ions. These results suggest that the 2'-OH of U(-1) does not interact with a metal ion. The contribution of the 2'-moiety of U(-1) is unperturbed by thio substitution at either of the nonbridging oxygens of the reactive phosphoryl group, providing no indication of a hydrogen bond between the 2'-OH and the nonbridging phosphoryl oxygens. In contrast, the 10(3)-fold catalytic advantage of 2'-OH relative to 2'-H is eliminated when the 3'-bridging oxygen is replaced by sulfur. As sulfur is a weaker hydrogen-bond acceptor than oxygen, this effect suggests a hydrogen-bonding interaction between the 2'-OH and the 3'-bridging oxygen. CONCLUSIONS: These results provide the first experimental support for the model in which the 2'-OH of U(-1) donates a hydrogen bond to the neighboring 3'-bridging oxygen, thereby stabilizing the developing negative charge on the 3'-oxygen in the transition state.     

A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme

BACKGROUND: The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. RESULTS: Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. CONCLUSIONS: These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.     

A role for a single-stranded junction in RNA binding and specificity by the Tetrahymena group I ribozyme

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.     

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme

BACKGROUND: Phosphoramidate oligonucleotide analogs containing N3'-P5' linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N-P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. RESULTS: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3'-P5' bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5' splicing reaction. CONCLUSIONS: Ribo-N3'-P5' phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein-RNA interactions.     

Probing the active site of a diels-alderase ribozyme by photoaffinity cross-linking

The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.     

Physical nature of intermolecular interactions within cAMP-dependent protein kinase active site: differential transition state stabilization in phosphoryl transfer reaction

The origin of enzyme catalytic activity may be effectively explored within the nonempirical theory of intermolecular interactions. The knowledge of electrostatic, exchange, delocalization, and correlation components of the transition state and substrates stabilization energy arising from each enzyme active site residue allows to examine the most essential physical effects involved in enzymatic catalysis. Consequently, one can build approximate models of the catalytic activity in a systematic and legitimate manner. Whenever the dominant role of electrostatic interactions is recognized or assumed, the properties of an optimal catalytic environment could be simply generalized and visualized by means of catalytic fields that, in turn, aids the design of new catalysts. Differential transition state stabilization (DTSS) methodology has been applied herein to the phosphoryl transfer reaction catalyzed by cAMP-dependent protein kinase (PKA). The MP2 results correlate well with the available experimental data and theoretical findings indicating that Lys72, Asp166, and the two magnesium ions contribute -22.7, -13.3, -32.4, and -15.2 kcal/mol to differential transition state stabilization, respectively. Although all interaction energy components except that of electron correlation contribution are meaningful, the first-order electrostatic term correlates perfectly with MP2 catalytic activity. Catalytic field technique was also employed to visualize crucial electrostatic features of an ideal catalyst and to compare the latter with the environment provided by PKA active site. The map of regional electronic chemical potential was used to analyze the unfavorable catalytic effect of Lys168. It was found that locally induced polarization of TS atoms thermodynamically destabilizes electrons, pulling them to regions displaying higher electronic chemical potential.     

Compound I of nitric oxide synthase: the active site protonation state

A quantum mechanical/molecular mechanical (QM/MM) study of the formation of the elusive active species Compound I (Cpd I) of nitric oxide synthase (NOS) from the oxyferrous intermediate shows that two protons have to be provided to produce a reaction that is reasonably exothermic and that leads to the appearance of a radical on the tetrahydrobiopterin cofactor. Molecular dynamics and energy considerations show that a possible source of proton is the water H-bond chain formed from the surface to the active site, but that a water molecule by itself cannot be the source of the proton; an H3O+ species that is propagated along the chain is more likely. The QM/MM calculations demonstrate that Cpd I and H2O are formed from the ferric-hydrogen peroxide complex in a unique heterolytic O-O cleavage mechanism. The properties of the so-formed Cpd I are compared with those of the known species of chloroperoxidase, and the geometry and spin densities are found to be compatible. The M?ssbauer parameters are calculated and may serve as experimental probes in attempts to characterize NOS Cpd I.     

Phenol-formaldehyde resin route to the synthesis of several iron group transition metal phosphides

Abstract

A series of iron, cobalt and nickel metal phosphides of chemical formula Fe_xP, Co₂P and Ni₂P with high specific surface areas of 331.1, 294.2 and 228.0 m² g^?1, respectively, was firstly synthesized by phenol-formaldehyde resin route. It was found that the as-prepared Co₂P and Ni₂P samples synthesized using phenol-formaldehyde resin as a carbon source showed much higher BET surface areas than those prepared using other carbon sources reported before, including cinnamic strong alkali anion exchange resin, p-phenylenediamine and hexamethylenetetramine. This phenol-formaldehyde resin route was proved to be as universal as traditional H₂ reduction method.     

Ring current effects in the active site of medium-chain Acyl-CoA dehydrogenase revealed by NMR spectroscopy

Medium-chain acyl-CoA dehydrogenase (MCAD) catalyzes the flavin-dependent oxidation of fatty acyl-CoAs to the corresponding trans-2-enoyl-CoAs. The interaction of hexadienoyl-CoA (HD-CoA), a product analogue, with recombinant pig MCAD (pMCAD) has been studied using (13)C NMR and (1)H-(13)C HSQC spectroscopy. Upon binding to oxidized pMCAD, the chemical shifts of the C1, C2, and C3 HD carbons are shifted upfield by 12.8, 2.1, and 13.8 ppm, respectively. In addition, the (1)H chemical shift of the C3-H is also shifted upfield by 1.31 ppm while the chemical shift of the C4 HD-CoA carbon is unchanged upon binding. These changes in chemical shift are unexpected given the results of previous Raman studies which revealed that the C3=C2-C1=O HD enone fragment is polarized upon binding to MCAD such that the electron density at the C3 and C1 carbons is reduced, not increased (Pellet et al. Biochemistry 2000, 39, 13982-13992). To investigate the apparent discrepancy between the NMR and Raman data for HD-CoA bound to MCAD, (13)C NMR spectra have been obtained for HD-CoA bound to enoyl-CoA hydratase, an enzyme system that has also previously been studied using Raman spectroscopy. Significantly, binding to enoyl-CoA hydratase causes the chemical shifts of the C1 and C3 HD carbons to move downfield by 4.8 and 5.6 ppm, respectively, while the C2 resonance moves upfield by 2.2 ppm, in close agreement with the alterations in electron density at these carbons predicted from Raman spectroscopy (Bell, A. F.; Wu, J.; Feng, Y.; Tonge, P. J. Biochemistry 2001, 40, 1725-33). The large increase in shielding experienced by the C1 and C3 HD carbons in the HD-CoA/MCAD complex is proposed to arise from the ring current field from the isoalloxazine portion of the flavin cofactor. The flavin ring current, which is only present when the enzyme is placed in an external magnetic field, also explains the differences in (13)C NMR chemical shifts for acetoacetyl-CoA when bound as an enolate to MCAD and enoyl-CoA hydratase and is used to rationalize the observation that the line widths of the C1 and C3 resonances are narrower when the ligands are bound to MCAD than when they are free in the protein solution.     

High-energy intermediate or stable transition state analogue: theoretical perspective of the active site and mechanism of beta-phosphoglucomutase

A recent crystal structure of beta-phosphoglucomutase from Lactococcus lactis is reported to contain a five-coordinate phosphorus with five oxygen ligands that is a high-energy reaction intermediate during the phosphoryl transfer in the isomerization of beta-glucose 1-phosphate to beta-glucose 6-phosphate. Subsequently, it has been suggested that this structure is a transition state analogue with a five-coordinate magnesium with two oxygen and three fluorine ligands. Two layer ONIOM(B3LYP:PM3MM) calculations have been performed to address the nature of this intermediate and the mechanism of the phosphoryl transfer. These calculations provide evidence that (1) the observed crystal structure is consistent with a five-coordinate magnesium (a stable transition state analogue), not a five-coordinate phosphorus (a phosphorane) as a high-energy intermediate, (2) the active site is stabilized by the extensive hydrogen-bonding network, (3) the transfer of the phosphoryl group proceeds through a moderate barrier (14 kcal mol-1) five-coordinate phosphorus without a stable phosphorane or metaphosphate intermediate, (4) this concerted transition state is directly coupled to a proton transfer from the oxygen of glucose to the carboxylic group of aspartate 10, and (5) a stable glucose 1,6-bis-phosphoglucose intermediate is formed.     

Efficient photocyclization of o-alkylbenzaldehydes in the solid state: direct observation of E-xylylenols en route to benzocyclobutenols

The photocyclization to benzocyclobutenols of o-alkyl aromatic aldehydes that are predestined for gamma-hydrogen abstraction is found to occur efficiently in the solid state; in contrast, solution-phase photolysis is known to afford a mixture of several products. It is shown that mesitaldehyde, which is a liquid, also undergoes efficient cyclization when subjected to photolysis as a solid inclusion complex. The marginal energy differences in the relative energies of the E-enols and the corresponding cyclobutenols in the case of cyano-substituted mesitaldehydes has permitted direct observation, for the first time, of the E-enols en route to benzocyclobutenols. The AM1 calculations suggest that the cyano-substitution causes intrinsic stabilization of the E-enols relative to the corresponding cyclobutenols, while the bromo groups do the opposite. The lack of observation of the red color in bromo- and formyl-substituted aldehydes is attributed to rapid cyclization of the E-enols to the their respective cyclobutenols even at low temperatures.     

Mechanism of chemical reactions in the active site of aspartate N-acetyltransferase NAT8L revealed by molecular modeling

The results of a computational study of the synthesis of a key brain metabolite, N-acetyl-l-aspartate, catalyzed by aspartate N-acetyltransferase, encoded by the NAT8L gene, are reported. The reaction Gibbs energy profiles were computed using molecular dynamics simulations with interaction potentials estimated on-the-fly by the quantum mechanics/molecular mechanics QM(PBE0/6-31G**)/MM(CHARMM) approach. The revealed reaction mechanism includes four elementary steps with corresponding activation energies not exceeding 14 kcal mol^?1     

A second transition state for chain transfer to monomer in olefin polymerization promoted by group 4 metal catalysts

beta-Hydrogen transfer (BHT) to monomer is the dominant chain termination pathway for olefin polymerization promoted by group 4 metal catalysts. The transition state (TSA) for BHT studied in earlier work is characterized by a strong metal-hydrogen interaction. Our theoretical study of a series of homogeneous single-site polymerization catalysts reveals the existence of a second transition state (TSC), competitive with TSA, which has no direct metal-hydrogen interaction and strongly resembles that for the main-group metal aluminum. The balance between the two reaction paths is sensitive to choice of metal and ligand structure.     

Electrophilic substitution of the acinitro group by an arylthio group in the dianion of 1,4-dinitrocyclooctatetraene

Conclusions The dianion of 1,4-dinitrocyclooctatetraene reacts with 2-nitrobenzenesulfenyl chloride by two parallel pathways: a) substitution of one of the acinitro groups by an arylthio group to form l-nitro-4-(2-nitrophenylthio)cyclooctatetraene and b) a two-electron oxidationreduction with the formation of 2,2'-dinitrodiphenyl disulfide. 4-Nitrobenzenesulfenyl chloride reacts with this dianion to give l-nitro-4-(4'-nitrophenylthio)cyclooctatetraene only upon activation with GaCl₃· 2,4-Dinitrobenzenesulfenyl chloride in the presence of GaCl3 reacts with this dianion only by the oxidation-reduction pathway.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 11, pp. 2590–2593, November, 1986.     

Carbon monoxide as an intrinsic ligand to iron in the active site of the iron-sulfur-cluster-free hydrogenase H2-forming methylenetetrahydromethanopterin dehydrogenase as revealed by infrared spectroscopy

The iron-sulfur-cluster-free hydrogenase Hmd (H(2)-forming methylenetetrahydromethanopterin dehydrogenase) from methanogenic archaea has recently been found to contain one iron associated tightly with an extractable cofactor of yet unknown structure. We report here that Hmd contains intrinsic CO bound to the Fe. Chemical analysis of Hmd revealed the presence of 2.4 +/- 0.2 mol of CO/mol of iron. Fourier transform infrared spectra of the native enzyme showed two bands of almost equal intensity at 2011 and 1944 cm(-)(1), interpreted as the stretching frequencies of two CO molecules bound to the same iron in an angle of 90 degrees . We also report on the effect of extrinsic (12)CO, (13)CO, (12)CN(-), and (13)CN(-) on the IR spectrum of Hmd.     

Protonation state of Asp120 in the binuclear active site of the metallo-beta-lactamase from Bacteroides fragilis

The determination of the protonation state of enzyme active sites may be crucial for the investigation of their mechanism of action. In the bizinc beta-lactamase family of enzymes, no consensus has been reached on the protonation state of a fully conserved amino acid present in the active site, Asp120. To address this issue, we carry out here density functional theory (DFT) calculations on large models (based on Bacteroides fragilis X-ray structure) which include the metal coordination polyhedron and groups interacting with it. Our calculations suggest that Asp120 is ionized. The relevance of this finding for site-directed mutagenesis experiments on the 120 position and on the mechanism of action is discussed.