The relation of rotational correlation times to adiabatic rotational barriers for alanine methyl groups in staphylococcal nuclease (SNase) is investigated. The hypothesis that methyl rotational barriers may be useful probes of local packing in proteins is supported by an analysis of ten X-ray crystal structures of SNase mutants. The barrier heights are consistent across a set of ten structures of a native SNase and mutants containing single-point mutations or single or double insertions, most in a ternary SNase complex. The barriers for different methyls have a range of 7.5 kcal/mol, which at 300 K would correspond to a five-order-of-magnitude range in correlation time. It is demonstrated that adiabatic rotational barriers can fluctuate significantly during an MD simulation of hydrated SNase, but that a Boltzmann weighted average is predictive of rotational correlation times determined from correlation functions. Even if a given methyl is on average quite sterically hindered, infrequently sampled low-barrier conformations may dominate the Boltzmann distribution. This result is consistent with the observed uniformity of NMR correlation times for (13)C-labeled methyls. The methyl barriers in simulation fluctuate on multiple time scales, which can make the precise relationship between methyl rotational correlation time and methyl rotation barriers complicated. The implications of these issues for the interpretation of correlation times determined from NMR and simulation are discussed.

Molecular dynamics simulations of an explicitly solvated cis-β-methylstyrene/chloroperoxidase-Compound I complex are performed to determine the cause of the high enantiospecificity of epoxidation. From the simulations, a two-dimensional free energy potential is calculated to distinguish binding potential wells from which reaction to 1S2R and 1R2S epoxide products may occur. Convergence of the free energy potential is accelerated with an adaptive biasing potential. Analysis of binding is followed by analysis of 1S2R and 1R2S reaction precursor structures in which the substrate, having left the binding wells, places its reactive double bond in steric proximity to the oxyferryl heme center. Structural analysis of binding and reaction precursor conformations is presented. We find that 1), a distortion of Glu(183) is important for CPO-catalyzed epoxidation as was postulated previously based on experimental results; 2), the free energy of binding does not provide significant differentiation between structures leading to the respective epoxide enantiomers; and 3), CPO's enantiospecificity toward cis-β-methylstyrene is likely to be caused by a specific group of residues which form a hydrophobic core surrounding the oxyferryl heme center.

Nucleophilic attack at an α,β‐unsaturated carbonyl moiety usually results in conjugate addition at the β‐carbon atom (1,4 or Michael addition) or, occasionally, in addition at the carbonyl carbon atom (1,2 addition). Recently, however, addition at the α‐carbon atom has been observed when strongly electron‐withdrawing groups are positioned at the carbon atom β relative to the carbonyl group [e.g., methyl 3,3‐bis(trifluoromethyl)propenoate (8) and ethyl 3‐(2,4‐dinitrophenyl)propenoate (24)]. We have performed theoretical calculations [HF/6−31+G(d) and B3LYP//HF/6−31+G(d)] for the addition of cyanide anion to model α,β‐unsaturated carbonyl compounds to determine trends in the regioselectivity with respect to properties of the substituents. The difference between the reaction barriers for α‐ vs. β‐addition decreases as the strength of electron‐withdrawing groups increases until, for sufficiently strong electron‐withdrawing groups, α‐addition becomes favored. The calculations are in agreement with the experimental results. We show that the regioselectivity can be predicted from partial atomic charges and properties of the frontier orbitals of the reactants. We also report new experimental evidence of α‐addition to polysubstituted cinnamates and cinnamaldehydes. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

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The mechanism of acid-catalyzed keto-enol tautomerization of β-cyclopentanedione (CPD) in solution is studied computationally. Reaction profiles are first calculated for a limited solvation environment using ab initio and density functional methods. Barrier heights for systems including up to one hydration shell of explicit water molecules depend strongly on the number of waters involved in proton transfer, and to a lesser but significant extent on the number of waters forming hydrogen bonds with waters in the proton transfer chain (each such water reduces the barrier by 4.4 kcal/mol on average). Barriers of 8-13 kcal/mol were obtained when a full or nearly full hydration shell was present, consistent with calculations for non-acid-catalyzed keto-enol tautomerization of related molecules. The presence of HOCl reduced the barrier by 4.5 kcal/mol viz-a-viz the gas phase, consistent with the well-known principle that keto-enol tautomerization can be acid or base catalyzed. Reaction was also modeled beginning with snapshots of reactant conformations taken from a 300 K molecular dynamics simulation of CPD, HOCl and 324 explicit waters. Reaction profiles were calculated at a QM/MM level with waters in the first hydration shell either fixed or energy minimized at each step along the reaction coordinate. A substantial variation in barrier height was observed in both cases, depending primarily on electrostatic interactions (hydrogen bonding) with first-hydration-shell waters and to a lesser extent on electrostatic interactions with more distant waters and geometric distortion effects. For the lowest barriers, the extent of barrier reduction by waters involved in proton transfer is consistent with the limited-solvation results, but further barrier reduction due to hydrogen-bonding to waters involved in proton transfer is not observed. It is postulated that this is because highly flexible structures such as extensive hydrogen bonding networks optimal for reaction are entropically disfavored and so may not contribute significantly to the observed reaction rate.