In order to interpret the observed isotopic fractionation it is necessaryto understand its relationship with the isotope effect(s) on steps that occur during the conversion of the initial reactant to the final product. We examine this relationship from the biochemical point of view and elaborate on the consequences of the assumptions that it is based on. We illustrate the discrepancies between theoretical and experimental interpretation of kinetic isotope effects on examples of dehalogenation reactions that occur at an aromatic carbon atom. The examples include 4-chlorobenzoyl-CoA dehalogenase-catalyzed conversion of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA, dehaloperoxidase-catalyzed conversion of 2,4,6-trichlorophenol to 2,6-dichloroquinone, and spontaneous hydrolysis of atrazine at pH 12. For this latter reaction we have measured the chlorine kinetic isotope effect and estimated its value theoretically at the DFT level of theory. Results of chlorine kinetic isotope effects suggest that the studied dechlorination reactions proceed in a single step with significant weakening of the carbon-chlorine bond in the transition state.

Microtubules (MTs) are structural components essential for cell morphology and organization. It has recently been shown that defects in the filament’s lattice structure can be healed to create stronger filaments in a local area and ultimately cause global changes in MT organization and cell mobility. The ability to break, causing a defect, and heal appears to be a physiologically relevant and important feature of the MT structure. Defects can be created by MT severing enzymes and are target sites for complete severing or for healing by newly incorporated dimers. One particular lattice defect, the MT lattice ‘‘seam” interface, is a location often speculated to be a weak site, a site of disassembly, or a target site for MT binding proteins. Despite seams existing in many MT structures, very little is known about the seam’s role in MT function and dynamics. In this study, we probed the mechanical stability of the seam interface by applying coarse-grained indenting molecular dynamics. We found that the seam interface is as structurally robust as the typical lattice structure of MTs. Our results suggest that, unlike prior results that claim the seam is a weak site, it is just as strong as any other location on the MT, corroborating recent mechanical measurements.

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The nickel(i) octaethylisobacteriochlorin anion ([OEiBCh-Ni]) is commonly used as a synthetic model of cofactor F from Methyl-Coenzyme M Reductase. In this regard, experimental studies show that [OEiBCh-Ni] can catalyze dehalogenation of aliphatic halides in DMF solution by a highly efficient S2 reaction. To better understand this process, we constructed theoretical models of the dehalogenation of chloromethane by a simple nickel(i) isobacteriochlorin anion and compared its reactivity with that of similar Ni complexes with other porphyrin-derived ligands: porphyrin, chlorin, bactreriochlorin, hexahydroporphyrin and octahydroporphyrin. Our calculations predict that all of the porphyrin derivative's model reactions proceed through low-spin complexes. Relative to the energy of the separate reactants the theoretical activation energies (free-energy barriers with solvation corrections) for the dehalogenation of chloromethane are similar for all of the porphyrin derivatives and range for the different functionals from 10-15 kcal mol for B3LYP to 5-10 kcal mol for M06-L and to 13-18 kcal mol for ωB97X-D. The relative free energies of the products of the dehalogenation step, L-Ni-Me adducts, have a range from -5 to -40 kcal mol for all functionals; generally becoming more negative with increasing saturation of the porphyrin ligand. Moreover, no significant differences in the theoretical chlorine kinetic isotope effect were discernable with change of porphyrin ligand.

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In crystal structures of the molybdenum complexes [(1,2,4-C5H2(t)Bu3)Mo(PMe3)2H3] (Cp(t)Bu3) and [(C5H(i)Pr4)Mo(PMe3)2H3] (Cp(i)Pr4), the Mo-bound hydrogen positions were resolved for Cp(t)Bu3, but not for Cp(i)Pr4. NMR experiments revealed the existence of an unknown mechanism for hydrogen atom exchange in these molecules, which can be "frozen out" for Cp(t)Bu3 but not for Cp(i)Pr4. Density functional theory calculations of the most stable conformations for both complexes in the gas phase and in a continuum solvent model indicate that the H's of the Cp(i)Pr4 complex are unresloved because of their disorder, which does not occur for Cp(t)Bu3. A corresponding examination of alternative rearrangement pathways shows that the rearrangements of the H's could occur by two mechanisms: parallel to the cyclopentadienyl (Cp) ring in a single step and perpendicular to the Cp ring in two steps. The parallel pathway is preferred for both molecules, but it has a lower energy barrier for Cp(i)Pr4 than for Cp(t)Bu3.

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.

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