A Transition-State Perspective on Y-Family DNA Polymerase η Fidelity in Comparison with X-Family DNA Polymerases λ and ß.

Deoxynucleotide misincorporation efficiencies can span a wide 104-fold range, from ∼10-2 to ∼10-6, depending principally on polymerase (pol) identity and DNA sequence context. We have addressed DNA pol fidelity mechanisms from a transition-state (TS) perspective using our "tool-kit" of dATP- and dGTP-ß,γ substrate analogues in which the pyrophosphate leaving group (p Ka4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p Ka4 values from 7.8 (CF2) to 12.3 [C(CH3)2]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( kpol) versus p Ka4, for Y-family error-prone pol η and X-family pols λ and ß to determine the extent to which different electrostatic active site environments alter kpol values. The apparent chemical rate constant ( kpol) is the rate-determining step for the three pols. The pols each exhibit a distinct catalytic signature that differs for formation of right (A·T) and wrong (G·T) incorporations observed as changes in slopes and displacements of the Brønsted lines, in relation to a reference LFER. Common to this signature among all three pols is a split linear pattern in which the analogues containing two halogens show kpol values that are systematically lower than would be predicted from their p Ka4 values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p Ka4 values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.

Computer simulations of the catalytic mechanism of wild-type and mutant ß-phosphoglucomutase.

ß-Phosphoglucomutase (ß-PGM) has served as an important model system for understanding biological phosphoryl transfer. This enzyme catalyzes the isomerization of ß-glucose-1-phosphate to ß-glucose-6-phosphate in a two-step process proceeding via a bisphosphate intermediate. The conventionally accepted mechanism is that both steps are concerted processes involving acid-base catalysis from a nearby aspartate (D10) side chain. This argument is supported by the observation that mutation of D10 leaves the enzyme with no detectable activity. However, computational studies have suggested that a substrate-assisted mechanism is viable for many phosphotransferases. Therefore, we carried out empirical valence bond (EVB) simulations to address the plausibility of this mechanistic alternative, including its role in the abolished catalytic activity of the D10S, D10C and D10N point mutants of ß-PGM. In addition, we considered both of these mechanisms when performing EVB calculations of the catalysis of the wild type (WT), H20A, H20Q, T16P, K76A, D170A and E169A/D170A protein variants. Our calculated activation free energies confirm that D10 is likely to serve as the general base/acid for the reaction catalyzed by the WT enzyme and all its variants, in which D10 is not chemically altered. Our calculations also suggest that D10 plays a dual role in structural organization and maintaining electrostatic balance in the active site. The correct positioning of this residue in a catalytically competent conformation is provided by a functionally important conformational change in this enzyme and by the extensive network of H-bonding interactions that appear to be exquisitely preorganized for the transition state stabilization.

Metal Ion Complexes of N,N'-Bis(2-Pyridylmethyl)-trans-1,2-Diaminocyclohexane-N,N'-Diacetic Acid, H2bpcd: Lanthanide(III)-bpcd2- Cationic Complexes.

The synthesis and characterization of N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetic acid (H2bpcd) cationic complexes of La(III), Nd(III), and Sm(III) are reported. The Ln(III)-bpcd2- complex ions, where bpcd2- stands for N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetate, were isolated as PF6- salts. These salts were characterized by elemental analysis, X-ray crystallography, IR, and 1H and 13C NMR spectroscopy. Binuclear [La2(bpcd)2(H2O)2]2+ crystallized from an aqueous solution in the monoclinic P21/c space group as a cocrystallate with Na2bpcd and NaPF6, nominally Na2.34[La1.22(C22H26N4O4)2(H2O)2][PF6]2·2H2O, with a = 11.3343(6) Å, b = 17.7090(9) Å, c = 15.0567(8) Å, ß = 110.632(3)°, and Z = 4 (Z' = 2). La is eight-coordinate with distorted dodecahedral coordination geometry provided by a N4O4 donor atom set. In addition to four N atoms from the bpcd2- ligand, La's coordination sphere includes O atoms from a water molecule and three acetate groups (one O atom from singly bound acetate and two O atoms from acetate groups that bridge the La centers). The 1H and 13C assignments for H2bpcd and the metal-bpcd2- complexes were made on the basis of 2D COSY and HSQC experiments, which established 1H-1H and 1H-13C correlations. The NMR spectral data were used to establish the symmetry of the cationic complexes present in aqueous solution. The data indicate that the La(III)-bpcd2- and Sm(III)-bpcd2- complexes are present in solution as a single species with C2 symmetry. The 1H NMR spectrum of [Nd(bpcd)]PF6 in D2O consists of eight considerably line-broadened, paramagnetic-shifted singlets. The ab initio quantum mechanical calculations at the PCM/MP2/SDD//HF/SDD level, which were established previously for determining isomerization energies for octahedral M(III)-bpad2- complex ions, were used to determine the relative free energies of the geometric isomers possible for eight- and nine-coordinate La(III)-bpcd2- cationic aqua complexes in aqueous solution, i.e., [La(bpcd)(H2O)2]+ and La(bpcd)(H2O)3]+.

Membrane-Anchored Cytochrome P450 1A2-Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices.

Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex.

Lipid molecules can induce an opening of membrane-facing tunnels in cytochrome P450 1A2.

Cytochrome P450 1A2 (P450 1A2, CYP1A2) is a membrane-bound enzyme that oxidizes a broad range of hydrophobic substrates. The structure and dynamics of both the catalytic and trans-membrane (TM) domains of this enzyme in the membrane/water environment were investigated using a multiscale computational approach, including coarse-grained and all-atom molecular dynamics. Starting from the spontaneous self-assembly of the system containing the TM or soluble domain immersed in randomized dilauroyl phosphatidylcholine (DLPC)/water mixture into their respective membrane-bound forms, we reconstituted the membrane-bound structure of the full-length P450 1A2. This structure includes a TM helix that spans the membrane, while being connected to the catalytic domain by a short flexible loop. Furthermore, in this model, the upper part of the TM helix interacts directly with a conserved and highly hydrophobic N-terminal proline-rich segment of the catalytic domain; this segment and the FG loop are immersed in the membrane, whereas the remaining portion of the catalytic domain remains exposed to aqueous solution. The shallow membrane immersion of the catalytic domain induces a depression in the opposite intact layer of the phospholipids. This structural effect may help in stabilizing the position of the TM helix directly beneath the catalytic domain. The partial immersion of the catalytic domain also allows for the enzyme substrates to enter the active site from either aqueous solution or phospholipid environment via several solvent- and membrane-facing tunnels in the full-length P450 1A2. The calculated tunnel dynamics indicated that the opening probability of the membrane-facing tunnels is significantly enhanced when a DLPC molecule spontaneously penetrates into the membrane-facing tunnel 2d. The energetics of the lipid penetration process were assessed by the linear interaction energy (LIE) approximation, and found to be thermodynamically feasible.

Metal Ion Complexes of N,N'-Bis(2-Pyridylmethyl)-trans-1,2-Diaminocyclohexane-N,N'-Diacetic Acid, H2bpcd: Cis/Trans Isomerization Equilibria.

The synthesis of N,N'-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane-N,N'-diacetic acid (H2bpcd) and its complexation of Ga(III) and Co(III) are reported. H2bpcd and the metal-bpcd(2-) complexes, isolated as hexafluorophosphate salts, were characterized by elemental analysis, X-ray crystallography, IR spectroscopy, and (1)H and (13)C NMR spectroscopy. [Ga(bpcd)]PF6, [Ga(C22H26N4O4)]PF6, crystallized in the orthorhombic space group Ibca, with a = 13.8975(7) Å, b = 15.0872(7) Å, c = 22.2418(10) Å, and Z = 8. Ga is coordinated in a distorted octahedral geometry provided by a N4O2 donor atom set with trans-monodentate acetate groups and cis-2-pyridylmethyl N atoms, i.e., the trans-O,O isomer. The diamagnetic [Co(bpcd)]PF6, [Co(C22H26N4O4)]PF6, also crystallized from solution in the Ibca space group as the trans-O,O isomer. The (1)H and (13)C assignments for H2bpcd and metal-bpcd(2-) complexes were made on the basis of 2D COSY and HSQC experiments, which were used to differentiate among three possible isomers, i.e., one cis (C1 symmetry) and two trans (C2 symmetry). NMR results indicate that the [Ga(bpcd)](+), [Co(bpcd)](+), and cis-O,O, cis-Npy,Npy-[Ga(bppd)](+) cations, where bppd(2-) stands for bis(2-pyridylmethyl)-1,3-diaminopropane diacetate, are present in solution as isomers with the same symmetry as observed in the solid state. The crystallographic data and the dramatic shift that occurs in the position of the cis/trans isomerization equilibria for the [Ga(bpad)](+) cations simply by increasing the number of bridging CH2 groups in the ligand's diamine backbone represent a unique opportunity to assess the accuracy of modern computational methods. The performance of several local density functionals using a pseudopotential-based SDD basis set was compared with the more rigorous HF and MP2 ab initio calculations. The SVWN5 and SV5LYP functionals provide significantly better Ga-O and Ga-N distances than the HF method or the nonlocal BLYP functional. However, to provide proper isomerization energies the pseudopotential-DFT calculations must be augmented by MP2 single-point energies and calculations of solvation free energies.

Quantum mechanical analysis of nonenzymatic nucleotidyl transfer reactions: kinetic and thermodynamic effects of ß-γ bridging groups of dNTP substrates.

Rate (k) and equilibrium (K) constants for the reaction of tetrahydrofuranol with a series of Mg(2+) complexes of methyl triphosphate analogues, CH3O-P(O2)-O-P(O2)-X-PO3(4-), X = O, CH2, CHCH3, C(CH3)2, CFCH3, CHF, CHCl, CHBr, CFCl, CF2, CCl2, and CBr2, forming phosphate diester and pyrophosphate or bisphosphonate in aqueous solution were evaluated by B3LYP/TZVP//HF/6-31G* quantum chemical calculations and Langevin dipoles and polarized continuum solvation models. The calculated log k and log K values were found to depend linearly on the experimental pKa4 of the conjugate acid of the corresponding pyrophosphate or bisphosphonate leaving group. The calculated slopes of these Brønsted linear free energy relationships were ßlg = -0.89 and ßeq = -0.93, respectively. The studied compounds also followed the linear relationship Δlog k = 0.8Δlog K, which became less steep, Δlog k = 0.6Δlog K, after the range of studied compounds was extended to include analogues that were doubly protonated on γ-phosphate, CH3O-P(O2)-O-P(O2)-X-PO3H2(2-). The scissile Pα-Olg bond length in studied methyl triphosphate analogues slightly increases with decreasing pKa of the leaving group; concomitantly, the CH3OPα(O2) moiety becomes more positive. These structural effects indicate that substituents with low pKa can facilitate both Pα-Olg bond breaking and the Pα-Onuc bond forming process, thus explaining the large negative ßlg calculated for the transition state geometry that has significantly longer Pα-Onuc distance than the Pα-Olg distance.

Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

Metal ion complexes of N,N'-bis(2-pyridylmethyl)-1,3-diaminopropane-N,N'-diacetic acid, H2bppd.

A higher yield synthesis of N,N'-bis(2-pyridylmethyl)-1,3-diaminopropane-N,N'-diacetic acid (H2bppd) and its complexation of trivalent metal ions (Al(III), Ga(III), In(III)) and selected lanthanides (Ln(III)) are reported. H2bppd and the metal-bppd(2-) complexes, isolated as hexafluorophosphate salts, were characterized by elemental analysis, mass spectrometry, IR, and (1)H and (13)C NMR spectroscopy. [Ga(bppd)]PF6, [Ga(C19H22N4O4)]PF6, was crystallized as colorless needles by slow evaporation from anhydrous methanol; its molecular structure was solved by direct X-ray crystallography methods. The compound crystallized in the monoclinic space group P21/c, with a = 9.6134(2) Å, b = 20.2505(4) Å, c = 11.6483(3) Å, ß = 97.520(1)(o), and Z = 4. Ga is coordinated in a distorted octahedral geometry provided by a N4O2 donor atom set with cis-monodentate acetate groups and cis-2-pyridylmethyl N atoms. Quantum mechanical calculations were performed for the three possible geometric isomers of a pseudo-octahedral metal-bppd(2-) complex with five different metal ions. The results indicate, that in aqueous solution, the stability of the trans-O,O isomer is similar to that of the cis-O,O; cis-Npy,Npy isomer but is greater than that of the trans-Npy,Npy isomer. Calculations for a six-coordinate La(III)-bppd(2-) complex converge to a structure with a very large Npy-La-Npy bond angle (146.4°), a high metal charge (2.28 au), and a high solvation free energy (-79.4 kcal/mol). The most stable geometric arrangement for bppd(2-) around the larger La(III) is best described as an open nestlike structure with space available for additional ligands. IR spectroscopy was used to investigate the nature of the H2bppd-metal complexes isolated in the solid state and the binding modes of the carboxylate functionalities. The spectra indicate that fully deprotonated [M(bppd)](+) complexes as well as partially protonated complexes [M(Hbppd)Cl](+) were isolated. The (1)H and (13)C assignments for H2bppd and metal-bppd(2-) complexes were made on the basis of 2D COSY, NOESY, and (1)H-(13)C HSQC experiments, which were used to differentiate among the cis (C1 symmetry) and the two trans (C2 symmetry) isomers.

Empirical valence bond simulations of the chemical mechanism of ATP to cAMP conversion by anthrax edema factor.

The two-metal catalysis by the adenylyl cyclase domain of the anthrax edema factor toxin was simulated using the empirical valence bond (EVB) quantum mechanical/molecular mechanical approach. These calculations considered the energetics of the nucleophile deprotonation and the formation of a new P-O bond in aqueous solution and in the enzyme-substrate complex present in the crystal structure models of the reactant and product states of the reaction. Our calculations support a reaction pathway that involves metal-assisted transfer of a proton from the nucleophile to the bulk aqueous solution followed by subsequent formation of an unstable pentavalent intermediate that decomposes into cAMP and pyrophosphate (PPi). This pathway involves ligand exchange in the first solvation sphere of the catalytic metal. At 12.9 kcal/mol, the barrier for the last step of the reaction, the cleavage of the P-O bond to PPi, corresponds to the highest point on the free energy profile for this reaction pathway. However, this energy is too close to the value of 11.4 kcal/mol calculated for the barrier of the nucleophilic attack step to reach a definitive conclusion about the rate-limiting step. The calculated reaction mechanism is supported by reasonable agreement between the experimental and calculated catalytic rate constant decrease caused by the mutation of the active site lysine 346 to arginine.

Catalytic effects of mutations of distant protein residues in human DNA polymerase ß: theory and experiment.

We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase ß (Pol ß). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol ß catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol ß using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol ß mutants.

An abridged transition state model to derive structure, dynamics, and energy components of DNA polymerase ß 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 ß), is performed within the framework of an electrostatic linear free energy response (EFER) model. The structure, dynamics, and energetics of pol ß-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 ß 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 ß fidelity.

CH···π interactions do not contribute to hydrogen transfer catalysis by glycerol dehydratase.

The role of the nonbonded CH···π interaction in the hydrogen abstraction from glycerol by the coenzyme B(12)-independent glycerol dehydratase (GDH) was examined using the QM/MM (ONIOM), MP2, and CCSD(T) methods. The studied CH···π interaction included the hydrogen atom of the -C(2)H(OH)- group of the glycerol substrate and the tyrosine-339 residue of the dehydratase. A contribution of this interaction to the stabilization of the transition state for the transfer of a hydrogen atom from the adjacent terminal C(1)H(2)(OH) group to cysteine 433 was determined by ab initio HF, MP2, and CCSD(T) calculations with the aug-cc-pvDZ basis set for the corresponding methane/benzene, methanol/phenol, and glycerol radical/phenol subsystems. The calculated CH···π distance, defined as the distance between the H atom and the center of the phenol ring, shortened from 2.62 to 2.52 Å upon going from the ground- to the transition-state of the GDH-catalyzed reaction. However, this shortening was not accompanied by the expected lowering of the CH···π interaction free energy. Instead, this interaction remained weak (about -1 kcal/mol) along the entire reaction coordinate. Additionally, the mutual orientation of the CH group and the phenol ring did not change significantly during the reaction. These results suggest that the phenol group of the tyrosine-339 does not contribute to lowering the activation barrier in the enzyme, but do not exclude the possibility that tyrosine 339 facilitates proper orientation of glycerol for the electrostatic catalysis, or inhibits side-reactions of the reactive glycerol radical intermediate.

Linear energy relationships for the octahedral preference of Mg, Ca and transition metal ions.

The geometry, atomic charges, force constants, and relative energies of the symmetric and distorted M(2+)(H(2)O)(4)(F(-))(2), M(3+)(H(2)O)(4)(F(-))(2), M(2+)(H(2)O)(3)(F(-))(2), and M(3+)(H(2)O)(3)(F(-))(2) metal complexes, M = Mg, Ca, Co, Cu, Fe, Mn, Ni, Zn, Cr, V, were calculated by using the B3LYP/TZVP density functional method in both gas phase and aqueous solution, modeled using the polarized continuum model. The deformation energy associated with moving one water ligand 12 degrees from the initial "octahedral" arrangement, in which all O-M-O, O-M-F, and F-M-F angles are either 90 degrees or 180 degrees, was calculated to examine the angular ligand flexibility. For all M(2+)(H(2)O)(4)(F(-))(2) complexes, this distortion increased the energy of the complex in proportion to the electrostatic potential-derived (ESP) charge of the metal, and in proportion to D(-10), where D is the distance from the distorted ligand to its closest neighbor. The octahedral stability was further examined by calculating the energies for the removal of a water ligand from the octahedral complex to form a square-pyramidal or trigonal-bipyramidal complex. The octahedral preference, defined as the negative of the corresponding binding energy of the ligand, was found to linearly correlate with the ESP charge of the metal in both the gas phase and aqueous solution. The obtained results indicate that quantum-mechanical covalent effects are of secondary importance for both the flexibility and the octahedral preference of M(2+)(H(2)O)(4)(F(-))(2) and M(3+)(H(2)O)(4)(F(-))(2) complexes. This conclusion and supporting data are important for the development of consistent molecular mechanical force fields of the studied metal ions.

Alkaline phosphatase inhibition by vanadyl-beta-diketone complexes: electron density effects.

A series of systematically modified vanadyl-beta-diketone complexes, VO(beta-diketone)(2), bearing substituent groups with different electron inductive properties were synthesized and evaluated as inhibitors against calf-intestine alkaline phosphatase (APase). A combination of biochemical and quantum mechanical techniques were employed to identify structure-activity relationships relevant for rational design of phosphatase inhibitors. Kinetic parameters and activation free energy, enthalpy, and entropy for calf-intestine APase-catalyzed dephosphorylation of para-nitrophenylphosphate were also determined along with the inhibition constants (K(i)) for the VO(beta-diketone)(2) complexes. Increased positive charge on the vanadyl group increases the inhibition potency of the complex while the absence of an available coordination site on the complex decreases its inhibition potency. These findings correlate well with the results of ab initio electron density calculations for the complexes.

[Validity of decision criteria for replacement of fillings]. / Die Validität von Entscheidungskriterien bei der Füllungserneuerung.

One of the main treatments in dental practice is the exchange of restorations due to secondary or residual caries. Thereby, only restorations indeed infected with secondary or residual caries should be renewed. The aim of the study was to check the validity of different criteria for the replacement of fillings. Three hundred seventeen replacements of dental restorations were evaluated retrospectively by using an examination form. Different clinical parameters were correlated with the finding of caries after removal of the old restoration. Clinical findings were differentiated between caries soft to probing, caries only stainable with caries detector and caries-free cavities. Sixty-seven percent of the cavities showed caries that could be probed, 16.1% were just stainable with caries detector and 17% were caries-free. In general, results of previous replacements of fillings were a valid criterion. Other indicators for caries-free cavities were properly placed fillings with a correctly reconstructed morphology, fillings without marginal defects, a low age of the filling and a positive impression of the patients' general hygiene. Indicators for cavities with secondary caries were marginal gaps, pain within the respective section of the jaw, a high number of filled surfaces and a bad impression of the general hygiene. Systematic diagnostic criteria should be adopted in decision making on replacement of fillings in order to avoid new restorations of caries-free cavities.

Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors.

The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.

Associative versus dissociative mechanisms of phosphate monoester hydrolysis: on the interpretation of activation entropies.

Phosphate monoester and anhydride hydrolysis is ubiquitous in biology, being involved in, amongst other things, signal transduction, energy production, and the regulation of protein function. Therefore, this reaction has understandably been the focus of intensive research. Nevertheless, the precise mechanism by which phosphate monoester hydrolysis proceeds remains controversial. Traditionally, it has been assumed and frequently implied that a near-zero activation entropy is indicative of a dissociative pathway. Herein, we examine free-energy surfaces for the hydrolysis of the methyl phosphate dianion and the methyl pyrophosphate trianion in aqueous solution. In both cases, the reaction can proceed through either compact or expansive concerted (A(N)D(N)) transition states, with fairly similar barriers. We have evaluated the activation entropies for each transition state and demonstrate that both associative and dissociative transition states have near-zero entropies of activation that are in good agreement with experimental values. Therefore, we believe that the activation entropy alone is not a useful diagnostic tool, as it depends not only on bond orders at the transition state, but also on other issues that include (but are not limited to) steric factors determining the configurational volumes available to reactants during the reaction, solvation and desolvation effects that may be associated with charge redistribution upon approaching the transition state and entropy changes associated with intramolecular degrees of freedom as the transition state is approached.

DNA polymerase beta catalytic efficiency mirrors the Asn279-dCTP H-bonding strength.

Ternary complexes of wild type or mutant form of human DNA polymerase beta (pol beta) bound to DNA and dCTP substrates were studied by molecular dynamics (MD) simulations. The occurrences of contact configurations (CC) of structurally important atom pairs were sampled along the MD trajectories, and converted into free-energy differences, DeltaG(CC). DeltaG(CC) values were correlated with the experimental binding and catalytic free energies for the wild type pol beta and its Arg183Ala, Tyr271Ala, Asp276Val, Lys280Gly, Arg283Ala, and Glu295Ala mutants. The correlation coefficients show that the strength of the H-bond between dCTP and Asn279 is a strong predictor of the mutation-induced changes in the catalytic efficiency of pol beta. This finding is consistent with the view that enzyme preorganization plays a major role in controlling DNA polymerase specific activity.

Nitramine anion fragmentation: a mass spectrometric and Ab initio study.

The fragment ion formation characteristics of the radical anions generated from hexahydro-1,3,5-trinitrotriazine (RDX) and its three nitroso metabolites were studied using GC/MS with negative chemical ionization (NCI) to understand the fragmentation mechanisms responsible for the formation of the most abundant ions observed in their NCI mass spectra. Ab initio and density functional theory calculations were used to calculate relative free energies for different fragment ion structures suggested by the m/z values of the most abundant ions observed in the NCI mass spectra. The NCI mass spectra of the four nitramines are dominated by ions formed by the cleavage of nitrogen-nitrogen and carbon-nitrogen bonds in the atrazine ring. The most abundant anions in the NCI mass spectra of these nitramines have the general formulas C(2)H(4)N(3)O (m/z 86) and C(2)H(4)N(3)O(2) (m/z 102). The analyses of isotope-labeled standards indicate that these two ions are formed by neutral losses that include two exocylic nitrogens and one atrazine ring nitrogen. Our calculations and observations of the nitramine mass spectra suggest that the m/z 86 and m/z 102 ions are formed from either the (M--NO)(-) or (M--NO(2))(-) fragment anions by a single fragmentation reaction producing neutral losses of CH(2)N(2)O or CH(2)N(2)O(2) rather than a set of sequential reactions involving neutral losses of HNO(2) or HNO and HCN.