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T C Bruice

Publications and source records attributed to T C Bruice.

At least 19 recordsLinked to original sources
PubMed2001

Dynamic structures of horse liver alcohol dehydrogenase (HLADH): results of molecular dynamics simulations of HLADH-NAD(+)-PhCH(2)OH, HLADH-NAD(+)-PhCH(2)O(-), and HLADH-NADH-PhCHO.

Molecular dynamics simulations of the oxidation of benzyl alcohol by horse liver alcohol dehydrogenase (HLADH) have been carried out. The following three states have been studied: HLADH.PhCH(2)OH.NAD(+) (MD1), HLADH.PhCH(2)O(-).NAD(+) (MD2), and HLADH.PhCHO.NADH (MD3). MD1, MD2, and MD3 simulations were carried out on one of the subunits of the dimeric enzyme covered in a 32-A-radius sphere of TIP3P water centered on the active site. The proton produced on ionization of the alcohol when HLADH.PhCH(2)OH.NAD(+) --> HLADH.PhCH(2)O(-).NAD(+) is transferred from the active site to solvent water via a hydrogen bonding network consisting of serine48 hydroxyl, ribose 2'- and 3'-hydroxyl groups, and Hist51. Hydrogen bonding of the 3'OH of ribose to Ile269 carbonyl maintains this proton in position to be transferred to water. Molecular dynamic simulations have been employed to track water1287 from the TIP3 water pool to the active site, thus exhibiting the mode of entrance of water to the active site. With time the water1287 accumulates in two different positions in order to accept the proton from the ribose 3'-OH and from His51. There can be identified two structural substates for proton passage. In the first substate the imidazole Ne2 of His51 is adjacent to the nicotinamide ribose C2'-OH and hydrogen bonding distances for proton transfer through the hydrogen bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...His51...OH(2) (path 1) average 2.0, 2.0, and 2.1 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A. The structure for path 1 is present 20% of the time span. And in the second substate, there are two possible proton passages: path 1 as before and path 2. Path 2 involves the hydrogen-bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...Ribose3'-OH...His51.OH(2) with the average bonding distances being 2.0, 2.0, 2.1, and 2.0 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A (20% probability of the time span), respectively. During the molecular dynamics simulation the NAD(+) ribose conformations have stabilized at the C2'-endo-C3'-exo or the C2'-endo conformations. With the C2'-endo conformation the first and second substates are able to persist for different time spans, while with the C2'-endo-C3'-exo conformation the only possible pathway involves the first substate. For both first and second substates the fluctuation of the distances between the ribose-OH protons and N epsilon 2 of His51 imidazole ring is partially contributed by the "windshield wiper" motion of the His51 imidazole ring. Since the imidazole of His-51 contributes only about 10-fold to activity, as estimated from the decrease in activity upon substitution with a Gln, there must be an alternate route for the proton to pass to solvent without going through this histidine. A third pathway involves ribose C3'-OH and Ile-269. In MD2, near attack conformers (NACs) for hydride transfer from PhCH(2)O(-) to NAD(+) represent approximately 60% of E.S conformers. The molecular dynamic study of MD3 at mildly basic pH reveals that reactive ground state conformers (NACs) for hydride transfer from NADH to PhCHO amount to 12 mol % of conformers. In MD3, anisotropic bending of the dihydronicotinamide ring of NADH (average value of alpha(c) = 4.0 degrees and alpha(n) = 0.5 degrees, respectively) is observed.

Alcohol Dehydrogenase↗
PubMed2001

Diphtheria toxin catalyzed hydrolysis of NAD(+): molecular dynamics study of enzyme-bound substrate, transition state, and inhibitor.

The mechanism of the diphtheria toxin-catalyzed hydrolysis of NAD(+) was investigated by quantum chemical calculations and molecular dynamics simulations. Several effects that could explain the 6000-fold rate acceleration (Delta Delta G(++) approximately 5 kcal/mol) by the enzyme were considered. First, the carboxamide arm of the enzyme-bound NAD(+) adopts a trans conformation while the most stable conformation is cis. The most stable conformation for the nicotinamide product has the amide carbonyl trans. The activation energy for the cleavage of the ribosidic bond is reduced by 2 kcal/mol due to the relaxation of this ground state conformational stress in the transition state. Second, molecular dynamics simulations to the nanosecond time range revealed that the carboxylate of Glu148 forms a hydrogen bond to the substrate's 2' hydroxyl group in E.S (approximately 17% of the time) and E.TS (approximately 57% of the time) complexes. This interaction is not seen in crystal structures. The ApUp inhibitor is held more tightly by the enzyme than the transition state and the substrate. Analysis of correlated motions reveals differences in the pattern of anticorrelated motions for protein backbone atoms when the transition state occupies the active site as compared to the E.NAD(+) complex.

Catalysis↗
PubMed2001

Inhibition of transcription factor-DNA complexes and gene expression by a microgonotropen.

Developing minor groove-binding drugs to selectively inhibit transcription factor (TF)/DNA interactions and accompanying gene expression is a current goal in drug development studies. Equipping minor groove-binding agents with positively charged, major groove-contacting side chains yields microgonotropens (MGTs). Previously, we demonstrated that MGTs were superior inhibitors of TF/DNA complexes in cell-free assays compared with "classical" groove binders, but MGTs showed limited ability to inhibit gene expression. To determine what chemical characteristics contribute to or improve activity, we evaluate five MGTs for their effectiveness in inhibiting TF complex formation and resultant transcription by using the c-fos serum response element (SRE) as a target. MGT L1 binds DNA via a bisbenzimidazole equipped with a tripyrrole moiety. It is compared with analog L2, which has been functionalized with propylamines on each of the three pyrroles. L2, which binds DNA at subpicomolar concentrations, was at least three orders of magnitude more potent than L1 at inhibiting TF binding to the c-fos SRE in cell-free assays. Unlike L1 and previous MGTs, L2 also inhibited endogenous c-fos expression in NIH 3T3 cells at micromolar levels. Structure/activity relationships suggest that, although the tripyrrole/polyamine functional group of L2 may be largely responsible for its inhibition of TF complexes in cell-free assays, its bisbenzimidazole moiety appears to impart improved cellular uptake and activity. These findings make L2 a promising lead candidate for future, rational MGT design.

3T3 Cells↗
PubMed2001

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase.

A molecular dynamics study was performed to compare the differences in the active-site dynamics of the wild-type and W137F mutant enzymes of 4-chlorobenzoyl-CoA dehalogenase. Only in the wild-type simulation are conformations formed between the catalytic Asp-145 and 4-chlorobenzoyl-CoA, which resemble the ab initio calculated gas-phase transition-state geometry. In the W137F simulation, the hydrogen bond formed between His-90 and Asp-145 persisted throughout the simulation, causing the carboxylate of Asp-145 to be distant from the benzoyl ring of 4-chlorobenzoyl-CoA. In both simulations, water molecules were able to diffuse into the active site of the enzymes. The trajectories provide insight into the routes that water may use to get into position for the hydrolysis portion of the dehalogenation reaction. In both simulations, the water molecule entering the active site forms a hydrogen bond with Asp-145.

Amino Acid Substitution↗
PubMed2001

Solid-phase synthesis of oligopurine deoxynucleic guanidine (DNG) and analysis of binding with DNA oligomers.

The first stepwise solid-phase synthesis of deoxynucleic guanidine (DNG), a positively charged DNA analog, using controlled pore glass as the solid support is reported. For the first time, purine bases have been incorporated into the DNG oligomer and DNG has been synthesized using a solid-phase method, proceeding in the 3'-->5' direction, that is compatible with the cleavage conditions used in the solid-phase synthesis of DNA. A DNG sequence containing a pentameric tract of adenosine nucleosides has been synthesized and the thermal denaturation temperature of its complexes with complementary thymidyl DNA oligomers was 79 degrees C. Binding of thymidyl DNA oligomers to adenyl DNG oligomers is 2:1, as seen in thymidyl and adenyl DNA triplexes. No binding of adenyl DNG with octameric cytidyl DNA was observed, indicating that the positive charge does not overcome base pairing fidelity.

Chromatography, High Pressure Liquid↗
PubMed2001

Double-stranded DNA binding characteristics and subcellular distribution of a minor groove binding diphenyl ether bisbenzimidazole.

The interactions of Hoechst 33377 (H1) with 20 different oligomeric duplexes have been investigated via spectrofluorometric titrations and/or thermal denaturation experiments. H1 is shown to form 2:1 complexes with dsDNA binding sites of at least four contiguous A/T base pairs. H1 is also shown to possess the rare ability to meaningfully distinguish between different A.T rich sequences. For example, the combined equilibrium constants for complexation of the oligomeric duplex 5'-GCAATTGC-3' (15) by H1 are found to be 110-fold greater than for the duplex 5'-GCTTAAGC-3' (16). It is believed that the 5'-TpA-3' dinucleotide step in 16 disrupts the rigid "A-tract" conformation of 15 and discourages minor groove binding by agents capable of recognizing longer dsDNA sequences. Molecular models are presented which elucidate the structure of the (H1)(2)-dsDNA minor groove complex. The two H1 molecules bind to an A/T rich sequence of 6 bp in a slightly staggered, side-by-side, and antiparallel arrangement. Evidence suggests that the piperazine rings of the H1 side-by-side complex are capable of resting in the minor groove of G/C base pairs. Fluorescence microscopy studies using NIH3T3 cells indicate that H1 is capable of traversing the cytoplasmic membrane and selectively localizing to nuclear DNA. H1 also demonstrated the ability to inhibit endogenous transcription of the c-fos gene in NIH3T3 cells at micromolar concentrations. Cytotoxicity studies employing the same cell type show H1 to possess an LD(50) of 3.5 microM.

3T3 Cells↗
PubMed2001

A theoretical examination of the acid-catalyzed and noncatalyzed ring-opening reaction of an oxirane by nucleophilic addition of acetate. Implications to epoxide hydrolases.

Ab initio and density functional calculations have been performed to gain a better understanding of the epoxide ring-opening reaction catalyzed by epoxide hydrolase. The S(N)2 reaction of acetate with 1S,2S-trans-2-methylstyrene oxide to provide the corresponding diol acetate ester was studied with and without general-acid catalysis. MP2 and DFT (B3LYP) calculations predict, for the noncatalyzed reaction, a central barrier of approximately 20-21 kcal/mol separating the reactants from products depending on which carbon center in the epoxide is undergoing attack. From these gas-phase reactions the immediate alkoxide products are not energetically far below their associated transition states such that the reaction is predicted to be endothermic. Inclusion of aqueous solvation effects via a polarizable continuum model predicts the activation barrier to increase by almost 10 kcal/mol due to the solvation of the acetate ion nucleophile. The activation barrier for the epoxide ring-opening reaction is reduced to approximately 10 kcal/mol when phenol, as the general-acid catalyst, is included in the gas-phase calculations. This is due to the immediate product being the neutral ester rather than the corresponding alkoxide. The transition state in the general-acid-catalyzed reaction is earlier than that for the noncatalyzed reaction and the reaction is highly exothermic. Molecular mechanics calculations of 1S,2S-trans-2-methylstyrene oxide in the active site of murine epoxide hydrolase show two possible binding conformations. Both conformers have the epoxide oxygen forming hydrogen bonds with the acidic hydrogens of the catalytic tyrosines (Tyr381 and Tyr465). These two conformations likely lead to different products since the nucleophile (Asp333-CO(2)(-)) is positioned to react with either carbon center in the epoxide.

Acetic Acid↗
PubMed2001

Recognition of nine base pairs in the minor groove of DNA by a tripyrrole peptide-Hoechst conjugate.

A tripyrrole peptide-Hoechst conjugate (FPH-1) has been designed which recognizes nine dA/dT base pair A/T rich dsDNA sequences at subnanomolar concentrations and complexes its targets at near diffusion controlled rates to form a fluorescent product. Spectrofluorometric titrations show the stoichiometry of the complex to be (FPH-1)(2):dsDNA. Spectrofluorometric titrations were also employed to determine the product of the equilibrium constant for complexation (K(1)K(2)) of dsDNA by two FPH-1 molecules for 35 different oligomeric duplexes. Single base pair mismatches in the FPH-1 binding site were found to cause significant decreases in K(1)K(2) of 18- to 2300-fold. Thermal denaturation experiments provided similar results. Arguments are presented which favor the structure of the (FPH-1)(2):dsDNA minor groove complex to involve the two FPH-1 molecules in a slightly staggered, side-by-side, and antiparallel arrangement such that the bis-benzimidazole moiety of one FPH-1 molecule lies adjacent to the tripyrrole moiety of the second FPH-1 molecule.

Base Pairing↗
PubMed2001

Catalytic mechanism of quinoprotein methanol dehydrogenase: A theoretical and x-ray crystallographic investigation.

The catalytic mechanism of the reductive half reaction of the quinoprotein methanol dehydrogenase (MDH) is believed to proceed either through a hemiketal intermediate or by direct transfer of a hydride ion from the substrate methyl group to the cofactor, pyrroloquinoline quinone (PQQ). A crystal structure of the enzyme-substrate complex of a similar quinoprotein, glucose dehydrogenase, has recently been reported that strongly favors the hydride transfer mechanism in that enzyme. A theoretical analysis and an improved refinement of the 1.9-A resolution crystal structure of MDH from Methylophilus methylotrophus W3A1 in the presence of methanol, reported earlier, indicates that the observed tetrahedral configuration of the C-5 atom of PQQ in that study represents the C-5-reduced form of the cofactor and lends support for a hydride transfer mechanism for MDH.

Alcohol Oxidoreductases↗
PubMed2000

The importance of reactant positioning in enzyme catalysis: a hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase.

Hybrid quantum mechanics/molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S(N)2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal/mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics/molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal/mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2-4 kcal/mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S(N)2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.

Acetates↗
PubMed2000

Synthesis of fluorescent microgonotropens (FMGTs) and their interactions with dsDNA.

A new class of microgonotropen compounds (FIMGTs), which fluoresce upon binding to dsDNA, is introduced. The FMGTs consist of a minor groove binding moiety based upon Hoescht 33258 covalently attached to a polyamine chain capable of interacting with the phosphodiester backbone of dsDNA. The interactions of FMGTs with dsDNA were investigated by fluorescence and UV spectroscopy. Several different dsDNA oligomers were studied to determine the effect of binding site sequence on stoichiometric and binding affinity. The FMGTs were found to bind a dsDNA oligomer that contained the sequence 5'-AATTT-3' with FMGT:dsDNA stoichiometrics equal to 2:1 or 3:1. Hoechst 33258 bound the same dsDNA oligomer with a 1:1 stoichiometry. The second and third order equilibrium constants for complexation were determined to be Log(K1K2) = 17.9 M(-2) and Log(K1K2K3) = 26.1 M(-3), respectively, for two of strongest binding FMGTs. From thermal melting experiments deltaTm for Hoechst 33258 was determined to be 10 degrees C while the deltaTm values for FMGTs ranged from 20-26 degrees C indicating the greater stability of the latter.

Bisbenzimidazole↗
PubMed2000

Alpha-ketoamides and alpha-ketocarbonyls: conformational analysis and development of all-atom OPLS force field.

The molecular structures and barriers for the internal rotation around the OC-CO single bond in four alpha-ketoamides and eight alpha-ketocarbonyls have been determined from the MP3/aug-cc-pVDZ and MP2/aug-cc-pVDZ calculations. Alpha-ketocarbonyls with non-bulky substituents adopt planar conformations with two carbonyl oxygens in s-trans arrangement. The s-cis conformation is significantly less stable due to the electrostatic repulsion between the two carbonyl groups. Primary and secondary alpha-ketoamides are planar when the substituent at the carbonyl carbon is hydrogen or methyl group but tertiary alpha-ketoamides adopt a conformation where the OC-CO unit is significantly bent. Based on current ab initio structural data, a set of OPLS-AA force field parameters has been derived. These parameters can be used for the modeling of a variety of alpha-ketoamide or alpha-ketocarbonyl containing drugs such as novel protease inhibitors or neuroregenerative polyketides.

Amides↗
PubMed2000

Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) modified oligonucleotides containing neutral urea linkages: effect of charge deletions on binding and fidelity.

A solid-phase synthesis for a DNA analogue with a mixed guanidinium and urea backbone is reported. This material is nearly identical in structure to deoxynucleic guanidine (DNG) but the neutral urea internucleoside linkages can be used to attenuate the overall positive charge on the oligomer. The opposite charge attraction between urea containing DNG oligomers (DNGUs) and complimentary DNA can be controlled so that the affinity of DNG for DNA does not overwhelm the base-pairing discrimination necessary for specific binding. Octameric DNGU containing between 1 and 3 urea substitutions covered the range between very tight and very weak bonding. Each deletion of a positive charge reduced the thermal denaturation temperature (Tm) by approximately 5 degrees C. Mismatches in the DNA oligomers reduced the Tm values by 3 to 5 degrees C for each of the DNGU oligomers. DNGUs were found to bind in a 2:1 fashion to complimentary DNA in the same manner as DNG.

Chromatography, High Pressure Liquid↗
PubMed2000

Generation and evaluation of putative neuroregenerative drugs. Part 1: virtual point mutations to the polyketide rapamycin.

This paper explores the use of computational methods to direct engineered biosynthesis based on the desired properties of the target compounds. The immunosuppressive properties of rapamycin are a result of the formation of the complex FKBP12-rapamycin-FRAP. Neuroregenerative properties are exhibited by the complex or complexes of rapamycin with FKBP proteins. Our objective has been to design biosynthetically available analogues of rapamycin that bind tightly to FKBP12 but not to FRAP. This has been carried out by successive single ketide deletions from the effector domain of rapamycin. The approach described here has yielded modified rapamycin analogues (RP2 and RP3) as targets for biosynthesis by modified polyketide synthases. RP2 and RP3 have an identical binding affinity (linear interaction energy calculation) to FKBP12 as rapamycin but little or no affinity for binding to FRAP.

Anti-Bacterial Agents↗
PubMed2000

Generation and evaluation of putative neuroregenerative drugs. Part 2: screening virtual libraries of novel polyketides which possess the binding domain of rapamycin.

The use of computational methods to direct engineered biosynthesis toward candidates based on the desired properties of the target compounds has been explored. The objective for this study has been the modification of rapamycin in order to eliminate its immunosuppressive activity and retain its neuroregenerative abilities. We have designed analogues of rapamycin which have truncated effector domains but retain the ability to bind to FKBP proteins, which is a prerequisite for the neuroregenerative abilities of the drugs. The procedures described here consist of the screening of large virtual libraries of molecules which retain the binding domain of rapamycin but in which different substitute ketide units replace the effector domain. These methods have provided analogues of rapamycin that cannot retain the immunosuppressive abilities of rapamycin, have a binding affinity to FKBP12 identical to that of rapamycin (by linear interaction energy calculations), and are suitable for synthesis by modified polyketide synthases.

Anti-Bacterial Agents↗