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1.  Metavanadate at the active site of the phosphatase VHZ 
Journal of the American Chemical Society  2012;134(35):14298-14301.
Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, crystal structures and NMR data suggest the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases due to its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species, and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex the central, planar VO3 moiety has only one apical ligand, the nucleophilic cysteine-95, and a gap in electron density between vanadium and sulfur. A computational analysis shows the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence shows can bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure.
doi:10.1021/ja305579h
PMCID: PMC3482157  PMID: 22876963
2.  On the Mechanism and Rate of Spontaneous Decomposition of Amino Acids 
The journal of physical chemistry. B  2011;115(46):13624-13632.
Spontaneous decarboxylation of amino acids is among the slowest known reactions; it is much less facile than the cleavage of amide bonds in polypeptides. Establishment of the kinetics and mechanisms for this fundamental reaction is important for gauging the proficiency of enzymes. In the present study, multiple mechanisms for glycine decomposition in water are explored using QM/MM Monte Carlo simulations and free energy perturbation theory. Simple CO2 detachment emerges as the preferred pathway for decarboxylation; it is followed by water-assisted proton transfer to yield the products, CO2 and methylamine. The computed free energy of activation of 45 kcal/mol, and the resulting rate-constant of 1 × 10−21 s−1, can be compared with an extrapolated experimental rate constant of ~2 × 10−17 s−1 at 25 °C. The half-life for the reaction is more than 1 billion years. Furthermore, examination of deamination finds simple NH3-detachment yielding α-lactone to be the favored route, though it is less facile than decarboxylation by kcal/mol. Ab initio and DFT calculations with the CPCM hydration model were also carried out for the reactions; the computed free energies of activation for glycine decarboxylation agree with the QM/MM result, while deamination is predicted to be more favorable. QM/MM calculations were also performed for decarboxylation of alanine; the computed barrier is 2 kcal/mol higher than for glycine in qualitative accord with experiment.
doi:10.1021/jp2081808
PMCID: PMC3241013  PMID: 21995727
3.  Selected AB4 2−/− (A = C, Si, Ge; B = Al, Ga, In) ions: a battle between covalency and aromaticity, and prediction of square planar Si in SiIn4 2−/−† 
CAl4 2−/− (D4h, 1A1g) is a cluster ion that has been established to be planar, aromatic, and contain a tetracoordinate planar C atom. Valence isoelectronic substitution of C with Si and Ge in this cluster leads to a radical change of structure toward distorted pentagonal species. We find that this structural change goes together with the cluster acquiring partial covalency of bonding between Si/Ge and Al4, facilitated by hybridization of the atomic orbitals (AOs). Counter intuitively, for the AAl4 2−/− (A = C, Si, Ge) clusters, hybridization in the dopant atom is strengthened from C, to Si, and to Ge, even though typically AOs are more likely to hybridize if they are closer in energy (i.e. in earlier elements in the Periodic Table). The trend is explained by the better overlap of the hybrids of the heavier dopants with the orbitals of Al4. From the thus understood trend, it is inferred that covalency in such clusters can be switched off, by varying the relative sizes of the AOs of the main element and the dopant. Using this mechanism, we then successfully killed covalency in Si, and predicted a new aromatic cluster ion containing a tetracoordinate square planar Si, SiIn4 2−/−.
doi:10.1039/c2cp41821e
PMCID: PMC3478443  PMID: 22868353
4.  How Metal Substitution Affects the Enzymatic Activity of Catechol-O-Methyltransferase 
PLoS ONE  2012;7(10):e47172.
Catechol-O-methyltransferase (COMT) degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II)), and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II) with Ca(II) leads to a complete deactivation of COMT; Fe(II) is slightly less than potent Mg(II), and Fe(III) is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II) bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II), whereas Fe(III) is unable to promote catalysis. In the case of Ca(II), a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III) it is the electronic effect, whereas in the case of Ca(II) it is instead the effect of suboptimal protein structure.
doi:10.1371/journal.pone.0047172
PMCID: PMC3466255  PMID: 23056605
5.  Rigid substructure search 
Bioinformatics  2011;27(9):1327-1329.
Motivation: Identifying the location of binding sites on proteins is of fundamental importance for a wide range of applications, including molecular docking, de novo drug design, structure identification and comparison of functional sites. Here we present Erebus, a web server that searches the entire Protein Data Bank for a given substructure defined by a set of atoms of interest, such as the binding scaffolds for small molecules. The identified substructure contains atoms having the same names, belonging to same amino acids and separated by the same distances (within a given tolerance) as the atoms of the query structure. The accuracy of a match is measured by the root-mean-square deviation or by the normal weight with a given variance. Tests show that our approach can reliably locate rigid binding scaffolds of drugs and metal ions.
Availability and Implementation: We provide this service through a web server at http://erebus.dokhlab.org.
Contact: dokh@unc.edu
doi:10.1093/bioinformatics/btr129
PMCID: PMC3138080  PMID: 21460026
6.  Polarization Effects for Hydrogen-Bonded Complexes of Substituted Phenols with Water and Chloride Ion 
Variations in hydrogen-bond strengths are investigated for complexes of nine para-substituted phenols (XPhOH) with a water molecule and chloride ion. Results from ab initio HF/6-311+G(d, p) and MP2/6-311+G(d, p)//HF/6-311+G(d, p) calculations are compared with those from the OPLS-AA and OPLS/CM1A force fields. In the OPLS-AA model, the partial charges on the hydroxyl group of phenol are not affected by the choice of para substituent, while the use of CM1A charges in the OPLS/CM1A approach does provide charge redistribution. The ab initio calculations reveal a 2.0-kcal/mol range in hydrogen-bond strengths for the XPhOH⋯OH2 complexes in the order X = NO2 > CN > CF3 > Cl > F > H >OH >CH3 > NH2. The pattern is not well-reproduced with OPLS-AA, which also compresses the variation to 0.7 kcal/mol. However, the OPLS/CM1A results are in good accord with the ab initio findings for both the ordering and range, 2.3 kcal/mol. The hydrogen bonding is, of course, weaker with XPhOH as acceptor, the order for X is largely inverted, and the range is reduced to ca. 1.0 kcal/mol. The substituent effects are found to be much greater for the chloride ion complexes with a range of 11 kcal/mol. For quantitative treatment of such strong ion-molecule interactions the need for fully polarizable force fields is demonstrated.
doi:10.1021/ct7001754
PMCID: PMC2996042  PMID: 21132092
7.  Why Urea Eliminates Ammonia Rather Than Hydrolyzes in Aqueous Solution 
A joint QM/MM and ab initio study on the decomposition of urea in the gas phase and in aqueous solution is reported. Numerous possible mechanisms of intramolecular decomposition and hydrolysis have been explored; intramolecular NH3-elimination assisted by a water molecule is found to have the lowest activation energy. The solvent effects were elucidated using the TIP4P explicit water model with free energy perturbation (FEP) calculations in conjunction with QM/MM Monte Carlo simulations. The explicit representation of the solvent was found to be essential for detailed resolution of the mechanism, identification of the rate-determining step, and evaluation of the barrier. The assisting water molecule acts as a hydrogen shuttle for the first step of the elimination reaction. The forming zwitterionic intermediate, H3NCONH, participates in 8–9 hydrogen bonds with water molecules. Its decomposition is found to be the rate-limiting step, and the overall free energy of activation for the decomposition of urea in water is computed to be ca. 37 kcal/mol; the barrier for hydrolysis by an addition/elimination mechanism is found to be ca. 40 kcal/mol. The differences in the electronic structure of the transition states of the NH3-elimination and hydrolysis were examined via natural bond order analysis. Destruction of urea’s resonance stabilization during hydrolysis via an addition/elimination mechanism, and its preservation in the rearrangement to the H3NCONH intermediate were identified as important factors in determining the preferred reaction route.
doi:10.1021/jp066478s
PMCID: PMC2995377  PMID: 17249815
8.  Origin of the Activity Drop with the E50D Variant of Catalytic Antibody 34E4 for Kemp Elimination 
In enzymes, multiple structural effects cooperatively lead to the high catalytic activity, while individually these effects can be small. The design of artificial enzymes requires the understanding and ability to manipulate such subtle effects. The 34E4 catalytic antibody, catalyzing Kemp elimination of 5-nitrobenzisoxazole, and its Glu50Asp (E50D) variant are the subject of the present investigation. This removal of only a methylene group yields an approximately 30-fold reduction in the rate for the catalyzed Kemp elimination. Here, the aim is to understand this difference in the catalytic performance. The mechanism of Kemp elimination catalyzed by 34E4 and the E50D mutant is elucidated using QM/MM Monte Carlo simulations and free energy perturbation theory. In both proteins, the reaction is shown to follow a single-step, concerted mechanism. In the mutant, the activation barrier rises by 2.4 kcal/mol, which corresponds to a 62-fold rate deceleration, in good agreement with the experimental data. The positions and functionality of the residues in the active site are monitored throughout the reaction. It is concluded that the looser contact with the base, shorter base-Asn58 contact, less favorable π-stacking with Trp91 in the transition state of the reaction, and different solvation pattern all contribute to the reduction of the reaction rate in the E50D variant of 34E4.
doi:10.1021/jp8076084
PMCID: PMC2702134  PMID: 19132861
9.  Catalytic Mechanism and Performance of Computationally Designed Enzymes for Kemp Elimination 
Journal of the American Chemical Society  2008;130(47):15907-15915.
A series of enzymes for Kemp elimination of 5-nitrobenzisoxazole have been recently designed and tested. In conjunction with the design process, extensive computational analyses were carried out to evaluate the potential performance of four of the designs, as presented here. The enzyme-catalyzed reactions were modeled using mixed quantum and molecular mechanics (QM/MM) calculations in the context of Monte Carlo (MC) statistical mechanics simulations. Free energy perturbation (FEP) calculations were used to characterize the free-energy surfaces for the catalyzed reactions as well as for reference processes in water. The simulations yielded detailed information about the catalytic mechanisms, activation barriers, and structural evolution of the active sites over the course of the reactions. The catalytic mechanism for the designed enzymes KE07, KE10(V131N), and KE15 was found to be concerted with proton transfer generally more advanced in the transition state than breaking of the isoxazolyl N-O bond. On the basis of the free-energy results, all three enzymes were anticipated to be active. Ideas for further improvement of the enzyme designs also emerged. On the technical side, the synergy of parallel QM/MM and experimental efforts in the design of artificial enzymes is well illustrated.
doi:10.1021/ja804040s
PMCID: PMC2680199  PMID: 18975945

Results 1-9 (9)