Biomedical subjects
Publications and source records attributed to T C Bruice.
Recent advances in computational methods and the availability of fast, affordable computers have made the modeling of enzymatic reactions practical. The remaining challenges include achieving the accuracy level at which thermodynamic parameters and kinetic constants for different substrates, mutant enzymes, or in the presence of allosteric effectors can be predicted quantitatively.
[reaction: see text] Replacement of the negatively charged phosphodiester linkages of RNA with positively charged guanidinium linkages provides the polycationic ribonucleic guanidine (RNG). RNG is anticipated to bind strongly to target DNA/RNA through the specific interactions of nucleobases and the attractive electrostatic interactions of backbones. Preparation of building blocks and the solid-phase synthesis of RNG are reported. Both trimeric and pentameric uridyl RNG have been synthesized.
The replacement of phosphodiester linkages of the polyanion DNA with S-methylthiourea linkers provides the polycation deoxyribonucleic S-methylthiourea (DNmt). Molecular dynamics studies to 1,220 ps of the hybrid triplex formed from octameric DNmt strands d(Tmt)8 with a complementary DNA oligomer strand d(Ap)8 have been carried out with explicit water solvent and Na+Cl- counterions under periodic boundary conditions using the CHARMM force field and the Ewald summation method. The Watson-Crick and Hoogsteen hydrogen-bonding patterns of the A/T tracts remained intact without any structural restraints for triplex structures throughout the simulation. The duplex portion of the triplex structure equilibrated at a B-DNA conformation in terms of the helical rise and other helical parameters. The dynamic structures of the DNmt x DNA x DNmt triplex were determined by examining histograms from the last 800 ps of the dynamics run. These included the hydrogen-bonding pattern (sequence recognition), three-centered bifurcating occurrences, minor groove width variations, and bending of tracts for the hybrid triplex structures. Together with the Watson-Crick hydrogen-bondings, the strong Hoogsteen hydrogen-bondings, the partially maintained three-centered bifurcatings in the Watson-Crick pair, and the medium-strength three-centered bifurcatings in the Hoogsteen pair suggest that the hybrid triplex is energetically favorable as compared to a duplex with similar base stacking, van der Waals interactions, and helical parameters. This is in agreement with our previously reported thermodynamic study, in which only triplex structures were observed in solution. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 20 degrees for the Watson-Crick portion of the averaged structure. Propeller twist (associated with three-centered hydrogen-bonding) up to -30 degrees, native to DNA AT base pairing, was also observed for the triplex structure. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3'-endo domain and C2'-endo domain for the DNmt strand. Water spines are observed in both minor and major grooves throughout the dynamics run. The molecular dynamics simulations of the structural properties of DNmt x DNA x DNmt hybrid triplex is compared to the DNG x DNA x DNG hybrid triplex (In DNG the -O-(PO2-)-O- linkers in DNA is replaced by -NH-C(=N+H2)-NH-).
The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase/reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II(-). Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5, 6-shift is 2.5 kcal/mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by approximately 6 kcal/mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of approximately 0.6 kcal/mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II(-) requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase.
A new type of microgonotropen that fluoresces upon binding to dsDNA has been synthesized. FMGT-1, an analogue of the minor groove binder Hoechst 33258, is functionalized with a polyamine chain capable of interacting with the phosphate backbone of DNA. Binding studies indicate that FMGT-1 binds more tightly to dsDNA than the parent compound Hoechst 33258.
A molecular dynamics study was performed on the DNA methyltransferase M. Hha I in a ternary complex with DNA and AdoMet in solution. Methylation involves addition of the Cys81 sulfhydryl anion to the 6-position of Cyt18, followed by a nucleophilic attack of the resultant carbanion at C5 on the AdoMet methyl group. It was found in this simulation that the distances between the sulfhydryl group (SG) of Cys81 to the C6 of Cyt18 (SG-C6) and methyl carbon (CH3) of AdoMet to the C5 of cytosine (CH3-C5) are dependent on the dihedral angle chi (O4'-C1'-N1-C2) of the nucleotide. When the chi angle of Cyt18 is low (< -80 degrees), the SG-C6 and CH3-C5 distances are large. A high chi angle (> -80 degrees) for the target cytosine residue reduces the distances for both SG-C6 and CH3-C5, and the angles formed between the cytosine ring and AdoMet correspond well to values for the transition state structures formed during methylation of cytosine from ab initio calculations. Two possible proton sources for protonation of N3 of the cytosine residue upon formation of the covalent intermediate were found in the simulation. The protonated amine group of AdoMet could provide a proton via a water bridge, or Arg163 could also be the source of the proton for N3 via a water bridge. The simulation provides insights into how the H5 of cytosine could go from the active site into solvent. Conserved residues Asn304 and Gln82 stabilize a water network within the active site of M. Hha I which provides a route for H5 to diffuse into bulk solvent. An initially distant water molecule was able to diffuse into the active site of the enzyme and replace a position of a crystallographic water molecule in close proximity to the C5 of cytosine. The movement of this water molecule showed that a channel exists between Gln82 and the AdoMet in M. Hha I which allows both water and protons to easily gain access to the active site of the enzyme.
Complementary short-strand DNA homooligomers and methylthiourea-linked homonucleosides associate and form triplexes in solution. The melting temperatures, Tm, the association and dissociation kinetic and thermodynamic parameters, and activation energies were determined by UV thermal analysis for the triplexes of short-strand DNA homooligomers [d(pA)10-d(pA)23] and poly(dA) with the methylthiourea-linked nucleoside [5'-NH3+-d(Tmt)4-T-OH [DNmt5]]. Circular dichroism studies show evidence of triple-helical association dependent on the length of the target homooligomer. The melting and cooling curves exhibit hysteresis behavior in the temperature range of 10-95 degrees C at 0.13 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (kon, Eon) and dissociation (koff, Eoff) were obtained. Tm decreases with the ionic strength and increases with increase in length of the monomers. The rate constants kon and koff at a given temperature (288 K-310 K) are dependent on the DNA strand length and also decrease and increase respectively with the ionic strength. The energies of activation for the association and dissociation processes are in the range of -18 to -38 kcal/mol and 3 to 18 kcal/mol, respectively. The equilibrium constant for the formation of the triplexes [5'-NH3+-d(Tmt)4-T-OH)2.d(pA)x, x = 10-23] is several orders of magnitude greater when compared with the triplexes of DNA. The number of base triplets in the nucleus of the DNmt2.DNA triple-helix (nucleation-zipping model) increases with decreased DNA oligomer length and with increased ionic strength. The values of DeltaH degrees calculated from the activation parameters are between -30 and -50 kcal/(mol base) and the values of DeltaG degrees are between -6 and -11 kcal/(mol base) for short-strand DNA.
The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.
The electronic nature of low-barrier hydrogen bonds (LBHBs) in enzymatic reactions is discussed based on combined low temperature neutron and x-ray diffraction experiments and on high level ab initio calculations by using the model substrate benzoylacetone. This molecule has a LBHB, as the intramolecular hydrogen bond is described by a double-well potential with a small barrier for hydrogen transfer. From an "atoms in molecules" analysis of the electron density, it is found that the hydrogen atom is stabilized by covalent bonds to both oxygens. Large atomic partial charges on the hydrogen-bonded atoms are found experimentally and theoretically. Therefore, the hydrogen bond gains stabilization from both covalency and from the normal electrostatic interactions found for long, weak hydrogen bonds. Based on comparisons with other systems having short-strong hydrogen bonds or LBHBs, it is proposed that all short-strong and LBHB systems possess similar electronic features of the hydrogen-bonded region, namely polar covalent bonds between the hydrogen atom and both heteroatoms in question.
The synthesis of mixed backbone oligodeoxynucleotides (18-mers) consisting of positively charged guanidinium linkages along with negatively charged phosphodiester linkages is carried out. The use of a base labile-protecting group for guanidinium linkage offers a synthetic strategy similar to standard oligonucleotide synthesis. The nuclease resistance of the oligodeoxyribonucleotides capped with guanidinium linkages at 5' and 3' ends are reported. The hybridization properties and sequence specificity of binding of these deoxynucleic guanidine/DNA chimeras with complementary DNA or RNA are described.
We have performed molecular dynamics (MD) calculations by using one of the recently solved crystal structures of a hammerhead ribozyme. By rotating the alpha, beta, gamma, delta, epsilon, and zeta torsion angles of the phosphate linkage of residue 17, the nucleobase at the cleavage site was slightly rotated out of the active site toward the solution. Unconstrained MD simulations exceeding 1 ns were performed on this starting structure solvated in water with explicit counter ions and two Mg2+ ions at the active site. Our results reveal that near attack conformations consistently were formed in the simulation. These near attack conformations are characterized by assumption of the 2'-hydroxyl to a near in-line position for attack on the -O-(PO2-)-O- phosphorous. Also during the time course of the MD study, one Mg2+ moved immediately to associate with a pro-R phosphate oxygen in the conserved core region, and the second Mg2+ remained associated with the pro-R oxygen on the phosphate linkage undergoing hydrolysis. These results are in accord with a one-metal ion mechanism of catalysis and give insight into the possible roles of many of the conserved residues in the ribozyme.
The synthesis of novel fully protected guanidinium linked dinucleoside for incorporation into oligonucleotide using solid-phase DNA synthesis methodology was developed. The three different protecting groups selected allow different deprotection conditions.
DNG is a DNA analogue wherein the negatively charged phosphate backbone linkages have been replaced by achiral positively charged guanidinium linkages and has high affinity for complimentary DNA. The synthesis of these compounds in solution phase has been severely limited due to diminishing yields and solubility limitations. For the first time, an efficient solid-phase synthesis for oligomeric is reported.
Scytalone dehydratase (EC 4.2.1.94) catalyzes the dehydration of two important intermediates in the biosynthesis of melanin, and it functions without metal ions or any cofactors. Using molecular orbital theory, we have examined the role of a critical water molecule in the mechanism of scytalone dehydratase. The water, together with an internal hydrogen bonding, contributes significantly to the stabilization of the transition state (or the enolate intermediate). The role of two active site tyrosines (Tyr-50 and Tyr-30) is (i) to hold the critical water in place so that it may stabilize the transition state without much structural rearrangement during the catalytic reaction, and (ii) to polarize the water, making it a better general acid. The stereochemistry of the scytalone dehydratase-catalyzed dehydration is also discussed.
The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303-CO2-general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.
The semiempirical PM3 method, calibrated against ab initio HF/6-31+G(d) theory, has been used to elucidate the reaction of 1, 2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO- with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.
Short strand DNA oligomers (A5G3A5, GA4G3A4G, G2A3G3A3G2, and G2A2G5A2G2) and the guanidinium (g) linked thymidyl nucleoside d(Tg)4-T-azido associate as triplexes. The melting temperatures, Tm, the association and dissociation kinetic and thermodynamic parameters and activation energies for the triplexes were determined by UV thermal analysis. The hypochromic shift and Tm for triplex formation increases with increase in concentration and decreases with the number of mismatches. The melting temperatures are between 35 and 55 degrees C in the range of ionic strength of 0.06-0.24 and decrease with increase in ionic strength at 100 deg/(ionic strength unit). The melting and cooling curves exhibit hysteresis behavior in the temperature range 5-95 degrees C at 0.2 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (k(on), E(on)) and dissociation (k(off), E(off)) processes were obtained. The second-order rate constants, k(on), for the triplex formation at 288 K are between 10 and 500 M(-2) s(-1). Values of k(on) increase with the decrease in the ionic strength. The first order rate constants for the dissociation, k(off), at 288 K are between 10(-6) and 40 x 10(-6) s(-1) and increase with increase in ionic strength. The energies of activation for the association and dissociation processes are in the range -22 to -9 kcal/mol and 8 to 29 kcal/mol, respectively. At 6.3 x 10(-5) M/base and at the physiological ionic strength (0.15-0.30) and below, the triplex structures formed with d(Tg)4-T-azido and A5G3A5 and GA4G3A4G have well-defined Tm values. The melting curves with G2A3G3A3G2 and G2A2G5A2G2 are very shallow with small hypochromic shifts denoting negligible binding at physiological ionic strength. Therefore, with the increase in the G content (mismatched base pairs) at a certain concentration (e.g., 6.3 x 10(-5) M/base), discrimination (change in fidelity) occurs in the formation and strength of binding of d(Tg)4-T-azido to d(pAn pGm) oligomers. The standard molar enthalpies for triplex formation have in general larger negative values at low ionic strength than at high ionic strength, indicating that at lower mu values the formation of triplexes of d(Tg)4-T-azido with d(pAn pGm) are more favorable. The values of deltaH(standard)(288) calculated from the activation parameters are between -17 and -49 kcal/mol, and the values of deltaG(standard)(288) are between -7.5 and -11.8 kcal/mol for A5G3A5, GA4G3A4G, G2A3G3A3G2, and G2A2G5A2G2, respectively. There is a linear relationship in the enthalpy-entropy compensation for the triplex melting thermodynamics.
The SN2 displacements of chloride ion from CH3Cl, C2H5Cl, and C2H4Cl2 by acetate and hydroxide ions have been investigated, using ab initio molecular orbital theory at the HF/6-31+G(d), MP2/6-31+G(d), and MP4/6-31+G(d) levels of theory. The central barriers (calculated from the initial ion-molecule complex) of the reactions, the differences of the overall reaction energies, and the geometries of the transition states are compared. Essential stereochemical changes before and after the displacement reactions are described for selected cases. The gas phase reactions of hydroxide with CH3Cl, C2H5Cl, and C2H4Cl2 have no overall barrier, but there is a small overall barrier for the reactions of acetate with CH3Cl, C2H5Cl, and C2H4Cl2. A self-consistent reaction field solvation model was used to examine the SN2 reactions between methyl chloride and hydroxide ion and between 1,2-dichloroethane and acetate in solution. As expected, the reactions in polar solvent have a large barrier. However, the transition state structures determined by ab initio calculations change only slightly in the presence of a highly polar solvent as compared with the gas phase. We also calibrated the PM3 method for future study of an enzymatic SN2 displacement of halogen.