Density functional theory geometry optimizations and reduction potential calculations are reported for all five known oxidation states of [Fe(4)S(4)(SCH(3))(4)](n)()(-) (n = 0, 1, 2, 3, 4) clusters that form the active sites of iron-sulfur proteins. The geometry-optimized structures tend to be slightly expanded relative to experiment, with the best comparison found in the [Fe(4)S(4)(SCH(3))(4)](2)(-) model cluster, having bond lengths 0.03 A longer on average than experimentally observed. Environmental effects are modeled with a continuum dielectric, allowing the solvent contribution to the reduction potential to be calculated. The calculated protein plus solvent effects on the reduction potentials of seven proteins (including high potential iron proteins, ferredoxins, the iron protein of nitrogenase, and the "X", "A", and "B" centers of photosystem I) are also examined. A good correlation between predicted and measured absolute reduction potentials for each oxidation state of the cluster is found, both for relative potentials within a given oxidation state and for the absolute potentials for all known couples. These calculations suggest that the number of amide dipole and hydrogen bonding interactions with the Fe(4)S(4) clusters play a key role in modulating the accessible redox couple. For the [Fe(4)S(4)](0) (all-ferrous) system, the experimentally observed S = 4 state is calculated to lie lowest in energy, and the predicted geometry and electronic properties for this state correlate well with the EXAFS and Mössbauer data. Cluster geometries are also predicted for the [Fe(4)S(4)](4+) (all-ferric) system, and the calculated reduction potential for the [Fe(4)S(4)(SCH(3))(4)](1)(-)(/0) redox couple is in good agreement with that estimated for experimental model clusters containing alkylthiolate ligands.

Cytochromes P450 3A4, 2D6, and 2C9 metabolize a large fraction of drugs. Knowing where these enzymes will preferentially oxidize a molecule, the regioselectivity, allows medicinal chemists to plan how best to block its metabolism. We present QSAR-based regioselectivity models for these enzymes calibrated against compiled literature data of drugs and drug-like compounds. These models are purely empirical and use only the structures of the substrates, in contrast to those models that simulate a specific mechanism like hydrogen radical abstraction, and/or use explicit models of active sites. Our most predictive models use three substructure descriptors and two physical property descriptors. Descriptor importances from the random forest QSAR method show that other factors than the immediate chemical environment and the accessibility of the hydrogen affect regioselectivity in all three isoforms. The cross-validated predictions of the models are compared to predictions from our earlier mechanistic model (Singh et al. J. Med. Chem. 2003, 46, 1330-1336) and predictions from MetaSite (Cruciani et al. J. Med. Chem. 2005, 48, 6970-6979).

No abstract

Aldehyde oxidase is a molybdenum hydroxylase that catalyzes the oxidation of aldehydes and nitrogen-containing heterocycles. The enzyme plays a dual role in the metabolism of physiologically important endogenous compounds and the biotransformation of xenobiotics. Using density functional theory methods, geometry optimization of tetrahedral intermediates of drugs and druglike compounds was examined to predict the likely metabolites of aldehyde oxidase. The calculations suggest that the lowest energy tetrahedral intermediate resulting from the initial substrate corresponds to the observed metabolite >or=90% of the time. Additional calculations were performed on a series of heterocyclic compounds where the products resulting from metabolism by xanthine oxidase and aldehyde oxidase differ in many instances. Again, the lowest energy tetrahedral intermediate corresponded to the observed product of aldehyde oxidase metabolism >or=90% for the compounds examined, while the observed products of xanthine oxidase were not well predicted.

The hammerhead ribozyme is an RNA molecule capable of self-cleavage at a unique site within its sequence. Hydrolysis of this phosphodiester linkage has been proposed to occur via an in-line attack geometry for nucleophilic displacement by the 2'-hydroxyl on the adjoining phosphorus to generate a 2',3'-cyclic phosphate ester with elimination of the 5'-hydroxyl group, requiring a divalent metal ion under physiological conditions. The proposed S(N)2(P) reaction mechanism was investigated using density functional theory calculations incorporating the hybrid functional B3LYP to study this metal ion-dependent reaction with a tetraaquo magnesium (II)-bound hydroxide ion. For the Mg(2+)-catalyzed reaction, the gas-phase geometry optimized calculations predict two transition states with a kinetically insignificant, yet clearly defined, pentacoordinate intermediate. The first transition state located for the reaction is characterized by internal nucleophilic attack coupled to proton transfer. The second transition state, the rate-determining step, involves breaking of the exocyclic P-O bond where a metal-ligated water molecule assists in the departure of the leaving group. These calculations demonstrate that the reaction mechanism incorporating a single metal ion, serving as a Lewis acid, functions as a general base and can afford the necessary stabilization to the leaving group by orienting a water molecule for catalysis.

Formate dehydrogenase (FDH) from Pseudomonas sp. 101 is a homodimeric enzyme that catalyzes oxidation of formate and the concomitant reduction of NAD+ to produce NADH and CO2. The dynamic motions and distances between functional groups in the active site of the formate dehydrogenase including the NAD+ cofactor and substrate have been investigated by molecular dynamics (MD) simulations, incorporating the substrate in one subunit (E·S) and the transition state in the other subunit (E·TS). Experimental kinetic isotope effects and calculated isotope effects are in excellent agreement, thus, the transition state in the enzymatic reaction is expected to closely resemble the structure determined by ab initio calculations. The simulation shows that the formate is held in place by persistent electrostatic interactions consisting of a bifurcated hydrogen bond between one formate oxygen and the guanido hydrogens of Arg284, and single hydrogen bonds from the other formate oxygen to a side chain amide proton of Asn146 and the backbone amide proton of Ile122. The conserved residues Arg284, Asn146, and Ile122 serve as pivots about which the C−H of formate swings to and from the C4N of NAD+. The C4N of NAD+ and the formate hydrogen are in position to react (near attack conformations, NACs) approximately 1.5% of the simulation time. An additional effect of the hydrogen bonding of the formate oxygens to Arg284, Asn146, and Ile122 is to prevent nucleophilic attack of the carboxylate on NAD+ to form an ester, which is the reaction favored in the gas phase. His332 plays a role in both the binding of formate and the generation of the near attack conformations. Further insight into the roles of other conserved amino acids (Pro97, Phe98, Asp308, and Ser334) at the catalytic site is provided. Comparisons of the electrostatic interactions at the active site of the enzyme with substrate and transition state show changes in hydrogen bonding due to charge differences; however, these changes are not consistent with the hypothesis of preferential stabilization of the transition state over the ground state.

No abstract

We have performed molecular dynamics (MD) calculations by using one of the recently solved crystal structures of a hammerhead ribozyme. By rotating the ␣, ␤, ␥, ␦, , and 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 Mg 2؉ 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-(PO 2 ؊ )-O-phosphorous. Also during the time course of the MD study, one Mg 2؉ moved immediately to associate with a pro-R phosphate oxygen in the conserved core region, and the second Mg 2؉ 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 hammerhead ribozyme is one of a small class of selfcleaving RNAs that catalyzes the hydrolysis of phosphate esters (1-4). This ribozyme consists of a conserved core of 15 nucleotides required for full activity formed by three base pairing stem regions (5). The secondary structure of the hammerhead ribozyme studied here is shown in Chart 1 by using the standard nomenclature (6), with the conserved bases shown in outlined letters and the cleavage site indicated by the arrow. Note that the hammerhead ribozyme structure of Chart 1 exists as two separate strands with functions of enzyme (E) and substrate (S) such that it cleaves in trans (4, 7).The hammerhead ribozyme catalyzes the chemically well studied RNA hydrolysis mechanism (8), which involves in-line nucleophilic attack of the 2Ј-hydroxyl on phosphate phosphorous at the 3Ј-position with elimination of 5Ј-hydroxyl and formation of a 2Ј,3Ј-cyclic phosphate ester with inversion of configuration at the phosphorus (Scheme 1 refs. 9-11).Hammerhead ribozyme solvolysis is first order in HO Ϫ and requires one or more divalent metal ions (10, 12, 13). Thus, two kinetically equivalent mechanisms may be considered. The first is that metal ligated hydroxide acts as a general base to initiate the reaction by forming the 2Ј-O Ϫ nucleophile at residue 17. A second possibility is specific base catalysis of 2Ј-O Ϫ formation by lyate HO Ϫ and participation of the metal ion elsewhere. The metal ion may participate in various ways. It has been shown that a metal ion must ligate to the pro-R oxygen of the scissile phosphate group undergoing reaction (14,15). By doing so, the negative charge on the phosphate is neutralized and facilitates nucleophilic attack on the phosphorus atom. This divalent metal ion or possibly an additional one can carry out the required stabilization of the developing negative charge on the 5Ј-leaving gr...