Cyclic oxyphosphoranes as model intermediates during splicing and cleavage of RNA: ab initio molecular orbital calculations on the conformational analysis.

Ab initio molecular orbital calculations have been carried out on hydrated adducts of methyl ethylene phosphate as a model intermediate during cleavage of RNA. Upon rotating the apical methoxyl group two kinds of stable conformers and two kinds of rotational transition states are located, the most stable conformation being gs-G where the dihedral angle between the apical methyl group and the basal ring oxygen is calculated to be 76 degrees. In this gs-G conformation one of the lone pairs on the apical oxygen is oriented antiperiplanar to the basal ring ester bond. The torsional energy required to rotate the methyl group about the phosphorus-apical oxygen bond leading to ts-C conformation, where the methyl group is eclipsed with the ring oxygen, is calculated to be 5.2 kcal/mol. Judging from the published substrate's coordinates in the RNase environment, the expected pentacoordinate-intermediate/transition state during the cleavage of RNA appears to be, in fact, the most stable gs-G conformation.     

Rate limiting P-O(5'') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate.

In order to examine the energetics in base-catalyzed hydrolysis of RNA, a tentative pentacoordinated intermediate (3) has been characterized by molecular orbital calculations. Ab initio studies at the level of 3-21G* indicate that, under the Cs symmetry restricted conditions, the P-O(2) bond possessing antiperiplanar (app) lone pair electrons (Ip) on the equatorial oxygen (O(3)) can be cleaved with almost no barrier (TS1 transition state; 0.08 kcal mol-1), from the pentacoordinated intermediate (3) of base-catalyzed hydrolysis of phosphate, compared to the P-O(5) bond (TS2 transition state; 28.9 kcal mol-1) which lacks app lp assistance from O(3). The dianionic intermediate, however, loses the TS1 transition state thus its property as an intermediate when the Cs restriction is removed. The analysis of the entire potential energy surface enables us to conclude that, in a related system examined by Lim and Karplus [1990) J. Am. Chem. Soc., 112, 5872-5873) for attack by OH- on ethylene phosphate monoanion, the TS1 transition state had also been lost and thus no intermediate had been found. These results further support our earlier conclusions (Taira et al. (1990) Protein Engineering, 3, 691-701) of rate limiting transition state possessing extended P-O(5') bond breaking character (the TS2 transition state) in the base-catalyzed hydrolysis of RNA. Finally, although the lack of 2',3' -migration of phosphate moieties in basic condition appears to be in accord with the short-lived intermediate, it really does not prove the absence of the intermediate. The detail will be discussed in the text.     

Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.     

Deprotonation stimulates productive folding in allosteric TRAP hammerhead ribozymes

Hammerhead ribozymes in crystals change conformation in response to deprotonation of the nucleophilic 2' OH, thereby aligning the hydroxyl for in-line displacement at the scissile phosphate. Published data do not address whether deprotonation affects folding in solution. Allosteric hammerhead "TRAPs," when activated by the appropriate oligonucleotide, show the expected log-linear relation between initial cleavage rate and pH. In contrast, attenuated TRAPs shows biphasic kinetics in which a rapid burst is followed by slow cleavage that is nearly independent of pH. Attenuated ribozymes are stimulated by urea at both low and high pH, confirming that rearrangement of secondary structure is rate-limiting for the attenuated ribozymes once they have folded. Plots of burst magnitude versus pH in the absence of urea show a sharp transition around pH 8.3, which is near the kinetic pKa for the cleavage reaction in Mg2+. Raising the pH after folding at pH 7.5 did not activate attenuated ribozymes even when the RNA was incubated at the elevated pH for extended periods prior to addition of Mg2+. In contrast, lowering the pH after folding at pH 9.5 rapidly re-established attenuation. Deprotonation of the ribozyme-substrate complex thus appears to alter the folding landscape such that a metastable "pre-activated" complex forms before the thermodynamically more stable attenuated state can be attained. From the initial partition into active and inactive conformers, we estimate that this deprotonation contributes approximately 1.2 kcal/mol toward stabilization of the active fold at a crucial step during folding of the TRAP. Assuming that the nucleophilic 2' OH is the relevant acid, its deprotonation would thus serve a dual role of favoring productive fold and enhancing the nucleophilicity of this oxygen.     

Anomeric specificity of L-fucose dehydrogenase: a stereochemical imperative in aldopyranose dehydrogenases?

A set of hypotheses is proposed that explains the anomeric specificity of aldopyranose dehydrogenases in terms of an evolutionarily selected function. The first hypothesis, based on stereoelectronic theory, argues that, in the "allowed" transition state for oxidation at the anomeric carbon, the two oxygens attached to the anomeric carbon each bear a lone pair of electrons antiperiplanar to the departing "hydride". The second hypothesis is that the dehydrogenase is functionally constrained to bind the anomer that has this arrangement of lone pairs in its lowest energy chair conformer. The anomeric specificity of L-fucose dehydrogenase is experimentally examined. The enzyme oxidizes preferentially the beta-anomer, consistent with the prediction made by these hypotheses. Available experimental data for other enzymes (D-glucose-6-phosphate dehydrogenase, D-glucose dehydrogenase, D-galactose dehydrogenase, D-abequose dehydrogenase, and D-arabinose dehydrogenase) are found to be also consistent with the proposed hypotheses.     

A comparison of the reaction mechanisms of iron- and manganese-containing 2,3-HPCD: an important spin transition for manganese

Homoprotocatechuate (HPCA) dioxygenases are enzymes that take part in the catabolism of aromatic compounds in the environment. They use molecular oxygen to perform the ring cleavage of ortho-dihydroxylated aromatic compounds. A theoretical investigation of the catalytic cycle for HPCA 2,3-dioxygenase isolated from Brevibacterium fuscum (Bf 2,3-HPCD) was performed using hybrid DFT with the B3LYP functional, and a reaction mechanism is suggested. Models of different sizes were built from the crystal structure of the enzyme and were used in the search for intermediates and transition states. It was found that the enzyme follows a reaction pathway similar to that for other non-heme iron dioxygenases, and for the manganese-dependent analog MndD, although with different energetics. The computational results suggest that the rate-limiting step for the whole reaction of Bf 2,3-HPCD is the protonation of the activated oxygen, with an energy barrier of 17.4 kcal/mol, in good agreement with the experimental value of 16 kcal/mol obtained from the overall rate of the reaction. Surprisingly, a very low barrier was found for the O-O bond cleavage step, 11.3 kcal/mol, as compared to 21.8 kcal/mol for MndD (sextet spin state). This result motivated additional studies of the manganese-dependent enzyme. Different spin coupling between the unpaired electrons on the metal and on the evolving substrate radical was observed for the two enzymes, and therefore the quartet spin state potential energy surface of the MndD reaction was studied. The calculations show a crossing between the sextet and the quartet surfaces, and it was concluded that a spin transition occurs and determines a barrier of 14.4 kcal/mol for the O-O bond cleavage, which is found to be the rate-limiting step in MndD. Thus the two 83% identical enzymes, using different metal ions as co-factors, were found to have similar activation energies (in agreement with experiment), but different rate-limiting steps.     

Arginine 127 stabilizes the transition state in carboxypeptidase

Crystallographic studies suggest that Arg-127 is a key amino acid in the hydrolysis of peptides and esters by carboxypeptidase A. The guanidinium group of Arg-127 is hypothesized to stabilize the oxyanion of the tetrahedral intermediate formed by the attack of water on the scissile carbonyl bond. We have replaced this amino acid in rat carboxypeptidase A1 with lysine (R127K), methionine (R127M), and alanine (R127A), in order to define the role of Arg-127 in carboxypeptidase catalyzed hydrolysis. The wild-type and mutant enzymes were expressed in yeast and purified. Kinetic studies show that Arg-127 substitution decreases kcat for both ester and amide substrates, whereas Km is relatively unchanged; for R127M and R127A this corresponds to a 6 kcal/mol decrease in transition state stabilization of the rate-limiting step. The binding affinity for the phosphonate transition state analog, Cbz-Phe-Ala(P)-OAla, was decreased by 5.4 kcal/mol, whereas binding affinity for the ground state inhibitor, DL-benzylsuccinic acid, was decreased by only 1.7 kcal/mol for R127M. Electrostatic calculations employing a finite difference solution to the Poisson-Boltzmann equation predict that the positive charge of Arg-127 should stabilize the transition state by 6-8 kcal/mol. Therefore, the experimental and theoretical data suggest that the primary role of Arg-127 is stabilization of the transition state through electrostatic interaction with the oxyanion.     

Mechanism for intein C-terminal cleavage: a proposal from quantum mechanical calculations

Inteins are autocatalytic protein cleavage and splicing elements. A cysteine to alanine mutation at the N-terminal of inteins inhibits splicing and isolates the C-terminal cleavage reaction. Experiments indicate an enhanced C-terminal cleavage reaction rate upon decreasing the solution pH for the cleavage mutant, which cannot be explained by the existing mechanistic framework. We use intein crystal structure data and the information about conserved amino acids to perform semiempirical PM3 calculations followed by high-level density functional theory calculations in both gas phase and implicit solvent environments. Based on these calculations, we propose a detailed “low pH” mechanism for intein C-terminal cleavage. Water plays an important role in the proposed reaction mechanism, acting as an acid as well as a base. The protonation of the scissile peptide bond nitrogen by a hydronium ion is an important first step in the reaction. That step is followed by the attack of the C-terminal asparagine side chain on its carbonyl carbon, causing succinimide formation and simultaneous peptide bond cleavage. The computed reaction energy barrier in the gas phase is ~33 kcal/mol and reduces to ~25 kcal/mol in solution, close to the 21 kcal/mol experimentally observed at pH 6.0. This mechanism is consistent with the observed increase in C-terminal cleavage activity at low pH for the cleavage mutant of the Mycobacterium tuberculosis RecA mini-intein.     

Theoretical mechanism of the formation of cholesteryl chloride from cholesterol and thionyl chloride

This work describes a theoretical approach to the substitution reaction mechanism involving the conversion of cholesterol to cholesteryl chloride. Two chlorosulfite ester molecules were formed as intermediates. An iso-steroid was found as the transition state. The final product was cholesteryl chloride and the side products were HCl and SO₂. Calculations were carried out at high level Hartree–Fock theory, using the 6–31G* basis set. From the electronic structure of the reactants, the most important physicochemical properties involved in the reaction pathway were used. Thus, to determine the participation of each molecule and to explain the mechanism of reaction; the total energy, HOMO and LUMO, atomic orbital contribution to frontier orbitals formation, electrostatic potentials, atomic charges, hardness and dipole moment were used. Characterization of intermediates and transition state was supported by their respective energy minima, fundamental frequencies and equilibrium geometry.Figure Synopsis of the reaction pathway. The reaction starts when the lone pair of the Ch oxygen interacts with the sulfur atom, releasing a chloride ion. As a result, the first intermediate is formed. Next, in the first intermediate the nucleophilic chloride ion bonds the electrophilic hydrogen atom, releasing HCl and yielding the second intermediate. In the second intermediate, the electrophilic H-atom from HCl bonds with the lone pair of the Cl atom adjacent to the sulfur atom, restoring HCl. Concurrently, SO₂ is liberated and causes the formation of the C3-C5 partial bond and breaking of the C5-C6 -bond leading to the transition state. In the transition state, the electrophilic H from HCl bonds with the Cl lone pair at C6-Cl, forming HCl again and leaving the C6 atom electron-deficient, which restores the C5-C6 -bond and breaks the C3-C5 partial bond. Finally, the electrophilic C3 atom and the nucleophilic Cl atom form a bond, yielding cholesteryl chloride. HCl and SO₂ are also formed as side products. The arrows show the rearrangement of electrons.     

Molecular modeling of the mechanochemical triggering mechanism for catalysis of carbon-cobalt bond homolysis in coenzyme B12

The possible contributions of the mechanochemical triggering effect to the enzymatic activation of the carbon-cobalt bond of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl) for homolytic cleavage have been studied by molecular modeling and semiempirical molecular orbital calculations. Classically, this effect has envisioned enzymatic compression of the axial Co-N bond in the ground state to cause upward folding of the corrin ring and subsequent sterically induced distortion of the Co-C bond leading to its destabilization. The models of this process show that in both methylcobalamin (CH3Cbl) and AdoCbl, compression of the axial Co-N bond does engender upward folding of the corrin ring, and that the extent of such upward folding is smaller in an analog in which the normal 5,6-dimethylbenzimidazole axial ligand is replaced by the sterically smaller ligand, imidazole (CH3(lm)Cbl and Ado(lm)Cbl). Furthermore, in AdoCbl, this upward folding of the corrin is accompanied by increases in the carbon-cobalt bond length and in the Co-C-C bond angle (which are also less pronounced in Ado(Im)Cbl), and which indicate that the Co-C bond is indeed destabilized by this mechanism. However, these effects on the Co-C bond are small, and destabilization of this bond by this mechanism is unlikely to contribute more than ca. 3 kcal mol(-1) towards the enzymatic catalysis of Co-C bond homolysis, far short of the observed ca. 14 kcal mol(-1). A second version of mechanochemical triggering, in which compression of the axial Co-N bond in the transition state for Co-C bond homolysis stabilizes the transition state by increased Co-N orbital overlap, has also been investigated. Stretching the Co-C bond to simulate the approach to the transition state was found to result in an upward folding of the corrin ring, a slight decrease in the axial Co-N bond length, a slight displacement of the metal atom from the plane of the equatorial nitrogens towards the "lower" axial ligand, and a decrease in strain energy amounting to about 8 kcal mol(-1) for both AdoCbl and Ado(Im)Cbl. In such modeled transition states, compression of the axial Co-N bond to just below 2.0 A (the distance subsequently found to provide maximal stabilization of the transition state by increased orbital overlap) required about 4 kcal mol(-1) for AdoCbl, and about 2.5 kcal mol(-1) for Ado(Im)Cbl. ZINDO/1 calculations on slightly simplified structures showed that maximal electronic stabilization of the transition state by about 10 kcal mol(-1) occurred at an axial Co-N bond distance of 1.96 A for both AdoCbl and Ado(Im)Cbl. The net result is that this type of transition state mechanochemical triggering can provide 14 kcal mol(-1) of transition state stabilization for AdoCbl, and about 15.5 kcal mol(-1) for the Ado(Im)Cbl, enough to completely explain the observed enzymatic catalysis. These results are discussed in the light of current knowledge about class I AdoCbl-dependent enzymes, in which the coenzyme is bound in its "base-off" conformation, with the lower axial ligand position occupied by the imidazole moiety of an active site histidine residue, and the class II enzymes, in which AdoCbl binds to the enzyme in its "base-on" conformation, and the pendent 5,6-dimethylbenzimidazole base remains coordinated to the metal during Co-C bond activation.     

Twisting of glycosidic bonds by hydrolases

Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bonds on hybrid Quantum Mechanics::Molecular Mechanics energy surfaces. Eight such maps were constructed, including one for α-maltose and three for different forms of methyl α-acarviosinide to provide energies for twisting of α-(1,4) glycosidic bonds. Maps were also made for β-thiocellobiose and for three β-cellobiose conformers having different glycon ring shapes to model distortions of β-(1,4) glycosidic bonds. Different GH families twist scissile glycosidic bonds differently, increasing their potential energies from 0.5 to 9.5 kcal/mol. In general, the direction of twisting of the glycosidic bond away from the conformation of lowest intramolecular energy correlates with the position (syn or anti) of the proton donor with respect to the glycon’s ring oxygen atom. This correlation suggests that glycosidic bond distortion is important for the optimal orientation of one of the glycosidic oxygen lone pairs toward the enzyme’s proton donor.     

Mechanism and energy diagram for O-O bond formation in the oxygen-evolving complex in photosystem II

The recent finding of a transition state with a significantly lower barrier than previously found, has made the mechanism for O-O bond formation in photosystem II much clearer. The full mechanism can be described in the following way. Electrons and protons are ejected from the oxygen-evolving complex (OEC) in an alternating fashion, avoiding unnecessary build-up of charge. The S0-S1 and S1-S2 transitions are quite exergonic, while the S2-S3 transition is only weakly exergonic. The strong endergonic S3-S4 transition is a key step in the mechanism in which an oxygen radical is produced, held by the dangling manganese outside the Mn3Ca cube. The O-O bond formation in the S4-state occurs by an attack of the oxygen radical on a bridging oxo ligand in the cube. The mechanism explains the presence of both a cube with bridging oxo ligands and a dangling manganese. Optimal orbital overlap puts further constraints on the structure of the OEC. An alternating spin alignment is necessary for a low barrier. The computed rate-limiting barrier of 14.7 kcal mol(-1) is in good agreement with experiments.     

A theoretical study of glucosamine synthase

Continuing our theoretical studies of glucosamine synthase catalysis, we have carried out MNDO and ab initio calculations of the first stage of the reaction, which involves the attack of a cysteine thiol group from the enzyme active site on the side chain carboxyamide group of glutamine, producing ammonia and thioester. The reactants were modelled by methyl mercaptate and acetamide, respectively. For two considered mechanisms of the reaction the energy surfaces were evaluated. Mechanism I, proposed by Chmara et al. (1985) involves the nucleophilic attack of a deprotonated thiol group on the carbonyl carbon atom. Mechanism II, postulated in our previous work (Tempczyk et al. 1989), assumes the concerted binding of the mercaptate sulphur to the carbonyl carbon and the sulfhydryl hydrogen to the amide nitrogen with simultaneous breaking of the S-H bond. The energy surface of mechanism I shows no minimum on the approach of the mercaptide anion towards the carbonyl carbon, which is also consistent with ab initio calculations in a 4-31 G basis set. Therefore, mechanism I seems to be unlikely. The same analysis of mechanism II shows that it leads to the desired products: methyl thioacetate and ammonia. The presence of a sulfhydryl hydrogen causes apparent pyramidicity of the amido nitrogen and lengthening of the C-N bond in the transition state, making conditions for the release of the ammonia molecule. The MNDO calculated energy barrier of the reaction is 50.1 kcal/mol and the approximate 4-31 G ab initio barrier (at the MNDO geometries of the substrate complex and the transition state) is 63 kcal/mol. The biggest energy contribution to the barrier comes from the breaking of the S-H bond, which also causes a large charge separation in the transition state. The latter affect may result in the stabilisation of the transition state in a real enzymatic environment when compared to the gas phase, e.g. by the interaction of the reacting center with a pair of oppositely charged amino acid side chains such as lysinium and glutamate (aspartate), which are present in the enzyme studied. To estimate the magnitude of this effect, molecular mechanics calculations were carried out on the reaction center at the transition state in our proposed model of the enzymatic active site. The site was supplemented by ammonium and acetate ion, which were to mimic the lysinium and glutamate/aspartate side chains. A transition state stabilization energy of 20 kcal/mol was obtained and this lowers the energy barrier to about 30 kcal/mol. This value is within the thermal energy range of an average protein and indicates that our mechanism is a possible route of glucosamine synthase catalysis. Offprint requests to: E. Borowski     

The tetrahymena ribozyme cleaves a 5'-methylene phosphonate monoester approximately 10(2)-fold faster than a normal phosphate diester: implications for enzyme catalysis of phosphoryl transfer reactions

Single-atom substrate modifications have revealed an intricate network of transition state interactions in the Tetrahymena ribozyme reaction. So far, these studies have targeted virtually every oxygen atom near the reaction center, except one, the 5'-bridging oxygen atom of the scissile phosphate. To address whether interactions with this atom play any role in catalysis, we used a new type of DNA substrate in which the 5'-oxygen is replaced with a methylene (-CH2-) unit. Under (kcat/Km)S conditions, the methylene phosphonate monoester substrate dCCCUCUT(mp)TA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts approximately 10(3)-fold faster than the analogous control substrates lacking the -CH2- modification. Experiments with DNA-RNA chimeric substrates reveal that the -CH2- modification enhances docking of the substrates into the catalytic core of the ribozyme by approximately 10-fold and stimulates the chemical cleavage by approximately 10(2)-fold. The docking effect apparently arises from the ability of the -CH2- unit to suppress inherently deleterious effects caused by the thymidine residue that immediately follows the cleavage site. To analyze the -O- to -CH2- modification in the absence of this thymidine residue, we prepared oligonucleotide substrates containing methyl phosphate or ethyl phosphonate at the reaction center, thereby eliminating the 3'-terminal TA4 nucleotidyl group. In this context, the -O- to -CH2-modification has no effect on docking but retains the approximately 10(2)-fold effect on the chemical step. To investigate further the stimulatory influence on the chemical step, we measured the "intrinsic" effect of the -O- to -CH2- modification in nonenzymatic reactions with nucleophiles. We found that in solution, the -CH2- modification stimulates chemical reactivity of the phosphorus center by <5-fold, substantially lower in magnitude than the stimulatory effect in the catalytic core of the ribozyme. The greater stimulatory effect of the -CH2- modification in the active site compared to in solution may arise from fortuitous changes in molecular geometry that allow the ribozyme to accommodate the phosphonate transition state better than the natural phosphodiester transition state. As the -CH2- unit lacks lone pair electrons, its effectiveness in the ribozyme reaction suggests that the 5'-oxygen of the scissile phosphate plays no role in catalysis via hydrogen bonding or metal ion coordination. Finally, we show by analysis of physical organic data that such interactions in general provide little catalytic advantage to RNA and protein phosphoryl transferases because the 5'-oxygen undergoes only a small buildup of negative charge during the reaction. In addition to its mechanistic significance for the Tetrahymena ribozyme reaction and phosphoryl transfer reactions in general, this work suggests that phosphonate monoesters may provide a novel molecular tool for determining whether the chemical step limits the rate of an enzymatic reaction. As methylene phosphonate monoesters react modestly faster than phosphate diesters in model reactions, a similarly modest stimulatory effect on an enzymatic reaction upon -CH2- substitution would suggest rate-limiting chemistry.     

A trimetal site and substrate distortion in a family II inorganic pyrophosphatase

We report the first crystal structures of a family II pyrophosphatase complexed with a substrate analogue, imidodiphosphate (PNP). These provide new insights into the catalytic reaction mechanism of this enzyme family. We were able to capture the substrate complex both by fluoride inhibition and by site-directed mutagenesis providing complementary snapshots of the Michaelis complex. Structures of both the fluoride-inhibited wild type and the H98Q variant of the PNP-Bacillus subtilis pyrophosphatase complex show a unique trinuclear metal center. Each metal ion coordinates a terminal oxygen on the electrophilic phosphate and a lone pair on the putative nucleophile, thus placing it in line with the scissile bond without any coordination by protein. The nucleophile moves further away from the electrophilic phosphorus site, to the opposite side of the trimetal plane, upon binding of substrate. In comparison with earlier product complexes, the side chain of Lys296 has swung in and so three positively charged side chains, His98, Lys205 and Lys296, now surround the bridging nitrogen in PNP. Finally, one of the active sites in the wild-type structure appears to show evidence of substrate distortion. Binding to the enzyme may thus strain the substrate and thus enhance the catalytic rate.     

Peptides containing the sulfonamide junction. 2. Structure and conformation of Z-Tau-Pro-D-Phe-NHiPr

The taurine (Tau) containing N-protected pseudotripeptide isopropylamide Z-Tau-Pro-D-Phe-NHiPr (1) has been specifically designed and synthesized as suitable model to test the ability of the sulfonamido group to participate as H-bond acceptor to a type II beta-turn and to get information on the preferred rotameric conformation around the S-N bond and the hybridization state of the nitrogen atom. The present structural investigation reveals that, although the sulfonamide junction is invariably folded in a gauche mode, the beta-turn structure, stabilized by the 4 --> 1 hydrogen bond, is not found in the crystal and the sulfonamido oxygen atoms are not involved in any intra- or intermolecular hydrogen-bond interaction. More than one conformer populates the CDCl(3) solution with only a minor contribution by the expected beta-turn. The Pro nitrogen is significantly pyramidalized and the nitrogen lone pair points in opposite direction to that of the Pro C(alpha)H bond thus adopting R chirality, in an arrangement practically identical to that found in the previously studied homochiral analogue Z-Tau-Pro-Phe-NHiPr.     

Linkage between substrate recognition and catalysis during cleavage of sarcin/ricin loop RNA by restrictocin

Restrictocin is a site-specific endoribonuclease that inactivates ribosomes by cleaving the sarcin/ricin loop (SRL) of 23S-28S rRNA. Here we present a kinetic and thermodynamic analysis of the SRL cleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocin binds to a 27-mer SRL model substrate (designated wild-type SRL) via electrostatic interactions to form a nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-type substrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atom at the scissile phosphate, reacts at a fully diffusion-limited rate (k2/K1/2 = 1.1 x 10(9) M-1 s-1). At pH 7.4, the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocin recognizes the SRL structure, which imparts 4.3 kcal/mol transition state stabilization relative to a single-stranded RNA. The two conserved SRL modules, bulged-G motif and GAGA tetraloop, contribute at least 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRL recognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-G motif to progress from the nonspecific ground state complex (E:S) to the higher-energy-specific complex (E.S) en route to the chemical transition state. Comparison of restrictocin with other ribonucleases revealed that restrictocin exhibits a 10(3)-10(6)-fold smaller ribonuclease activity against single-stranded RNA than do the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findings show how structural features of the SRL substrate facilitate catalysis and provide a mechanism for distinguishing between cognate and noncognate RNA.     

An abridged transition state model to derive structure, dynamics, and energy components of DNA polymerase β fidelity

We show how a restricted reaction surface can be used to facilitate the calculation of biologically important contributions of active site geometries and dynamics to DNA polymerase fidelity. Our analysis, using human DNA polymerase beta (pol β), is performed within the framework of an electrostatic linear free energy response (EFER) model. The structure, dynamics, and energetics of pol β-DNA-dNTP interactions are computed between two points on the multidimensional reaction free energy surface. "Point 1" represents a ground state activation intermediate (GSA), which is obtained by deprotonating the terminal 3'OH group of the primer DNA strand. "Point 2" is the transition state (PTS) for the attack of the 3'O(-) (O(nuc)) on the P(α) atom of dNTP substrate, having the electron density of a dianionic phosphorane intermediate. Classical molecular dynamics simulations are used to compute the geometric and dynamic contributions to the formation of right and wrong O(nuc)-P chemical bonds. Matched dCTP·G and mismatched dATP·G base pairs are used to illustrate the analysis. Compared to the dCTP·G base pair, the dATP·G mismatch has fewer GSA configurations with short distances between O(nuc) and P(α) atoms and between the oxygen in the scissile P-O bond (O(lg)) and the nearest structural water. The thumb subdomain conformation of the GSA complex is more open for the mismatch, and the H-bonds in the mispair become more extended during the nucleophilic attack than in the correct pair. The electrostatic contributions of pol β and DNA residues to catalysis of the right and wrong P-O(nuc) bond formation are 5.3 and 3.1 kcal/mol, respectively, resulting in an 80-fold contribution to fidelity. The EFER calculations illustrate the considerable importance of Arg183 and an O(lg)-proximal water molecule to pol β fidelity.     

Tetraethylorthosilicate as molecular precursor to the formation of amorphous silica networks. A DFT-SCRF study of the base catalyzed hydrolysis

Quantum chemical calculations using density functional theory have been carried out to investigate two chemical pathways for the last step of the hydrolysis of tetraethylorthosilicate (TEOS) in basic catalyzed environment. The two models that are introduced in this study depend on the number of water molecules involved at the base catalyzed hydrolysis. Solution equilibrium geometries of the molecules involved in the transition states, reactants and product complexes of the two chemical pathways were fully optimized at B3LYP level of theory with the standard 6-31+G(d) basis set, modeling solvent effects using a polarizable continuum solvation model (PCM). Both models predict relative low activation energies. However, the model with two water molecules seems to be more adequate to describe the basic hydrolysis. A natural bond orbital (NBO) analysis seems to show that the proton transfer from water to ethoxy group would occur through a large hyperconjugative interaction, LP(O) → σ*(O-H), which is related to the nonbonding oxygen lone pair orbital from ethoxy group with the vicinal σ*(O-H) anti bonding orbital O-H of a water molecule.     

PvuII-endonuclease induces structural alterations at the scissile phosphate group of its cognate DNA

We investigated the PvuII endonuclease with its cognate DNA by means of molecular dynamics simulations. Comparing the complexed DNA with a reference simulation of free DNA, we saw structural changes at the scissile phosphodiester bond. At this GpC step, the enzyme induces the highest twist and axial rise, inclination is increased and the minor groove widened. The distance between the scissile phosphate group and the phosphate group of the following thymine base is shortened significantly, indicating a substrate-assisted catalysis. A feasible reason for this vicinity is the catalytically important amino acid residue lysine 70, which bridges the free oxygen atoms of the successive phosphate groups. Due to this geometry, a compact reaction pocket is formed where a water molecule can be held, thus bringing the reaction partners for hydrolysis into contact. The O1-P-O2 angle of the scissile nucleotide is decreased, probably due to a complexation of the negative oxygen atoms through protein and solvent contacts.